Compounds, compositions, and methods for treatment of diseases involving acidic or hypoxic diseased tissues

ABSTRACT

Compounds for treatment of diseases having acidic or hypoxic diseased tissues and pharmaceutical compositions comprising the compounds, as well as methods for making and using the compounds and compositions.

FIELD OF THE INVENTION

The present invention relates to therapeutic compounds andpharmaceutical compositions for treatment of diseases involving acidicor hypoxic diseased tissues, including cancer, cardiovascular diseasessuch as stroke and myocardial infarction, and long-termneurodegenerative diseases, as well as to methods for their use andmanufacture.

BACKGROUND OF THE INVENTION

Hypoxia and acidosis are physiological markers of many diseaseprocesses, including cancer. In cancer, hypoxia is one mechanismresponsible for development of an acid environment within solid tumors.As a result, hydrogen ions must be removed from the cell (e.g., by aproton pump) to maintain a normal pH within the cell. As a consequenceof this export of hydrogen ions, cancer cells have an increased pHgradient across the cell membrane lipid bilayer and a lower pH in theextracellular milieu when compared to normal cells.

Cancer is a group of diseases characterized by aberrant control of cellgrowth. The annual incidence of cancer is estimated to be in excess of1.6 million in the United States alone. While surgery, radiation,chemotherapy, and hormones are used to treat cancer, it remains thesecond leading cause of death in the U.S. It is estimated that about600,000 Americans will die from cancer each year.

Treatment of cancer in humans by systemic administration ofpharmaceutical agents often functions by slowing or terminating theuncontrolled replication that is a characteristic of cancer cells. Oneclass of such agents is DNA repair inhibitors. Such slowing ortermination, however, affects not only the replication of cancer cellsbut also the replication of non-cancerous cells, which leads to thewell-known undesirable side effects of such cancer treatment. It wouldbe highly desirable to selectively deliver such agents to the targetcancer cells and minimize or avoid the side effects caused by systemicadministration.

The PARP enzyme family, including poly(ADP-ribose) polymerase (PARP-1)and the related enzymes PARP-2 and PARP-3 (collectively “PARP”), areimportant elements in the repair of DNA single-strand breaks,particularly by the base excision repair pathway. If single-strandbreaks are not repaired prior to DNA replication, then double strandbreaks may form. Cells with increasing numbers of double strand breaksbecome more dependent on other repair pathways, such as homologousrecombination, and (if single strand breaks continue unrepaired) thecells die.

Inhibitors of PARP have been developed and continue to be developed asanti-cancer agents. Because these inhibitors prevent the repair of DNAsingle-strand breaks, they have a variety of roles in treatment ofcancer. (See, e.g., Nicola J. Curtin and Ricky A. Sharma—“PARPInhibitors for Cancer Treatment”, Cancer Drug Discovery and Development,vol 83, Humana Press 2015.)

PARP inhibitors can be used for treatment of forms of cancer that aremore dependent on PARP than normal cells (so-called “PARP-sensitive”cancers). For example, patients with homologous recombination deficiency(HRD), such as that involving the BRCA1 and BRCA2 genes, arebeneficially treated with a PARP inhibitor, either as monotherapy or incombination with other agents. Since DNA repair and survival of PARPinhibited cells are heavily dependent on HR, patients carrying BRCArelated mutations (who exhibit HRD) are well suited to treatment withPARP inhibitors.

Although PARP inhibitors are useful in the treatment of cancer, thecompounds also exhibit side effects. PARP side effects, which includeserious hematologic and gastrointestinal adverse reactions andpotentially fatal acute myeloid leukemia, are highly undesirable.

Other DNA repair inhibiting cancer therapeutics would be expected tosimilarly exhibit unwanted side effects when administered systemically.Such other DNA repair inhibiting cancer therapeutics include thosetargeting the protein kinase ataxia-telangiectasia mutated (ATM), theATM-Rad3-related protein kinase (ATR), and the nuclear serine/threonineprotein kinase DNA-PK. Cancer therapy would benefit significantly ifcompounds acting by these mechanisms could be delivered selectively tocancer calls and thus avoid undesired effects on normal cells.

Moreover, PARP inhibitors have potential for utility in treatment ofother diseases besides cancer, including cardiovascular and inflammatorydiseases, through possible roles for PARP in functions other than DNArepair, such as involvement in regulating the mitochondria-to-nucleustranslocation of apoptosis-inducing-factor (AIF) or involvement inregulating the expression of proteins implicated in inflammation. (See,e.g., Pacher and Szabo—“Role of Poly(ADP-ribose) polymerase 1 (PARP-1)in Cardiovascular Diseases: The Therapeutic Potential of PARPInhibitors, Cardiovasc Drug Rev. 2007; 25(3): 235-260). Preferentialdelivery of PARP inhibitors to these diseased tissues would likewise bebeneficial.

SUMMARY

The present disclosure provides, inter alia, a compound of Formula (I):R8-Q-R7   (I)or a pharmaceutically acceptable salt thereof, wherein constituentvariables are defined herein.

Broadly speaking, the invention provides (1) compounds comprising atherapeutic molecule (e.g., R⁷) and a pH-sensitive (or pH-dependent)peptide (e.g., R⁸) linked together, such as by a disulfide-containinglinker or other linker which would be cleaved in the intracellularenvironment, (2) pharmaceutical compositions comprising these compoundsand a pharmaceutically-acceptable carrier, (3) methods of using thesecompounds and compositions in the treatment of diseases and conditionsin humans and other mammals involving acidic and/or hypoxic cells, and(4) methods of making the compounds and compositions, and intermediatesuseful in said methods.

The present disclosure further provides a pharmaceutical compositioncomprising a compound of the disclosure, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient.

The present disclosure also provides methods of treating diseases orconditions involving acidic or hypoxic diseased tissues by administeringto a human or other mammal in need of such treatment a therapeuticallyeffective amount of a compound of the disclosure. The disclosure alsoprovides methods of reducing bone marrow toxicity associated withadministration of an ionizing radiation or cytotoxic agent, whichcomprises administering to a human or other mammal atherapeutically-effective amount of a compound of the disclosure incombination with the ionizing radiation or cytotoxic agent.

The present disclosure also provides uses of the compounds describedherein in the manufacture of a medicament for use in therapy. Thepresent disclosure also provides the compounds described herein for usein therapy.

The present disclosure also provides methods for synthesizing thecompounds of the disclosure and intermediates useful in these methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the tumor growth delay of R⁸H-15 and Example 12 inBRCA^(−/−) Mice.

FIG. 2 shows survival of R⁸H-15 and Example 12 in BRCA^(−/−) Mice.

FIG. 3 shows the tumor growth delay of R⁸H-16 and Example 18 inBRCA^(−/−) Mice.

FIG. 4 shows the effect of 9-Day intraperitoneal administration ofExample 18 and R⁸H-16 on tumor PARylation in a murine DLD-1 BRCA2^(−/−)xenograft model.

FIG. 5 shows the PARylation in bone marrow cells following intravenousadministration of Example 12 or oral administration of R⁸H-15 incombination with oral administration of temozolomide (TMZ) to nude mice.

FIG. 6 shows PARylation in bone marrow cells following intraperitonealadministration of Example 18 or R⁸H-16 to nude mice.

DETAILED DESCRIPTION

Provided herein is a compound of Formula (I):R8-Q-R7   (I)or a pharmaceutically acceptable salt thereof, wherein:

R⁷ is a peptide;

R⁸ is selected from the group consisting of:

Q is selected from the group consisting of

R¹, R², R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹² are each independentlyselected from H, C₁₋₄ alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1), wherein said C₁₋₄ alkyl, C₁₋₄ alkenyl, C₆₋₁₀aryl, and 5-10 membered heteroaryl are each optionally substituted with1, 2, or 3 substituents independently selected from halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R¹ and R² together with the carbon atom to which they are attachedform a C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R¹ and R³ together with the carbon atom to which they are attachedform a C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R³ and R⁴ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R⁵ and R⁶ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

R¹³ is H or C₁₋₆ alkyl;

A is H or C₁₋₄ alkyl;

is C₆₋₁₀ aryl or 5-10 membered heteroaryl; wherein the 5-10 memberedheteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4ring-forming heteroatoms independently selected from N, O, and S;

[N, O, S] is NH, O, or S;

[N, O] is NH or O;

[C, N, O] is CR^(X)R^(Y), NH, or O;

each R^(X) and R^(Y) are independently selected from H and C₁₋₄ alkyl;

[AA]x is a peptide that may be cleaved by enzymatic action;

S1 is

each R^(a), R^(b), R^(c), and R^(d) are independently selected from H,C₁₋₄ alkyl, OR^(a2), CO₂R^(a2), and OC(═O)R^(a2), wherein said C₁₋₄alkyl is optionally substituted with OR^(a2), CO₂R^(a2), andOC(═O)R^(a2);

R^(a1), R^(b1), R^(c1), and R^(d1) are each independently selected fromH, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl, OH, CN, NO₂,and CO₂CH₃; wherein said C₁₋₆ alkyl and C₂₋₆ alkenyl are each optionallysubstituted with OH, CN, NO₂, or CO₂CH₃;

R^(a2) is H or C₁₋₄ alkyl; and

n is 0 or 1.

In some embodiments, the lefthand side of Q attaches to R⁸ and therighthand side of Q attaches to R⁷.

In some embodiments, a sulfur atom of the disulfide moiety of Q is partof a cysteine residue of R⁷.

Suitable peptides for use as R⁷ are described, for example, in U.S. Pat.Nos. 8,076,451 and 9,289,508 (which are incorporated herein by referencein their entirety), although other peptides capable of such selectiveinsertion could be used. Other suitable peptides are described, forexample, in Weerakkody, et al., PNAS 110 (15), 5834-5839 (Apr. 9, 2013),which is also incorporated herein by reference in its entirety. Withoutbeing bound by theory, it is believed that the R⁷ peptide reversiblyfolds and inserts across cell membranes in response to pH changes. TheR⁷ peptide can target acidic tissue and selectively translocate polar,cell-impermeable molecules across cell membranes in response to lowextracellular pH. In some embodiments, R⁷ is a peptide capable ofselectively delivering R₈Q-across a cell membrane having an acidic orhypoxic mantle having a pH less than about 6.0. In some embodiments, R⁷is a peptide capable of selectively delivering R₈Q-across a cellmembrane having an acidic or hypoxic mantle having a pH less than about6.5. In some embodiments, R⁷ is a peptide capable of selectivelydelivering R₈Q-across a cell membrane having an acidic or hypoxic mantlehaving a pH less than about 5.5. In some embodiments, R⁷ is a peptidecapable of selectively delivering R₈Q-across a cell membrane having anacidic or hypoxic mantle having a pH between about 5.0 and about 6.0.

In some embodiments, R⁷ is attached to Q through a cysteine residue ofR⁷. In some embodiments, the sulfur atom of the cysteine residue canform part of the disulfide bond of the disulfide bond-containing linker.

In some embodiments, R⁷ is a peptide comprising at least one of thefollowing sequences:

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG, (SEQ ID NO. 2; Pv2)AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, and (SEQ ID NO. 3; Pv3)ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG; (SEQ ID NO. 4; Pv4)Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG; and (SEQ ID No. 5; Pv5)AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC;wherein R⁷ is attached to Q through a cysteine residue of R⁷.

In some embodiments, R⁷ is a peptide comprising at least one of thefollowing sequences:

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG, (SEQ ID NO. 2; Pv2)AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, and (SEQ ID NO. 3; Pv3)ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG,wherein R⁷ is attached to Q through a cysteine residue of R⁷.

In some embodiments, R⁷ is a peptide comprising the sequence

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG.

In some embodiments, R⁷ is a peptide comprising the sequence

(SEQ ID NO. 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.

In some embodiments, R⁷ is a peptide comprising the sequence

(SEQ ID NO. 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG.

In some embodiments, R⁷ is a peptide comprising the sequence

(SEQ ID NO. 4; Pv4) Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG.

In some embodiments, R⁷ is a peptide comprising the sequence

(SEQ ID NO. 5; Pv5) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC.

In some embodiments, R⁷ is a peptide consisting of the sequence

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG.

In some embodiments, R⁷ is a peptide consisting of the sequence

(SEQ ID NO. 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.

In some embodiments, R⁷ is a peptide consisting of the sequence

(SEQ ID NO. 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG.

In some embodiments, R⁷ is a peptide consisting of the sequence Ac—

(SEQ ID NO. 4; Pv4) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG.

In some embodiments, R⁷ is a peptide consisting of the sequence

(SEQ ID NO. 5; Pv5) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC.

In some embodiments, R⁷ is a peptide comprising at least one sequenceselected from SEQ ID NO: 6 to SEQ ID NO: 311 as shown in Table 1.

In some embodiments, R⁷ is a peptide consisting of a sequence selectedfrom SEQ ID NO: 6 to SEQ ID NO: 311 as shown in Table 1.

TABLE 1 Additional R⁷ Sequences SEQ ID NO. Sequence 6AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG 7AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 8GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 9AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 10AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 11GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 12ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 13ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 14AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 15AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 16AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT 17ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG 18ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG 19GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 20AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT 21AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT 22AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT 23AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGT 24AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT 25AAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT 26GEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 27GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 28GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 29GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 30GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG 31AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT 32DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 33DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGCT 34DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 35DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 36AEQNPIYWARYADFLFTTPLLLLDLALLVDADET 37AEQNPIYFARYADWLFTTPLLLLDLALLVDADEGT 38AEQNPIYFARYADFLFTTPLLLLDLALLWDADET 39 AKEDQNPYWARYADWLFTTPLLLLDLALLVDG40 ACEDQNPYWARYADWLFTTPLLLLDLALLVDG 41 AEDQNPYWARYADWLFTTPLLLLDLALLVDCG42 AEDQNPYWARYADWLFTTPLLLLELALLVECG 43 AKEDQNPYWRAYADLFTPLTLLDLLALWDG 44ACEDQNPYWRAYADLFTPLTLLDLLALWDG 45 ACDDQNPWRAYLDLLFPTDTLLLDLLW 46TEDADVLLALDLLLLPTTFLWD 47 AEQNPIYWARYADWLFTTPL 48 AEQNPIYWARYADWLFTTPCL49 ACEQNPIYWARYADWLFTTPL 50 AEQNPIYFARYADWLFTTPL 51KEDQNPWARYADLLFPTTLAW 52 ACEDQNPWARYADLLFPTTLAW 53ACEDQNPWARYADWLFPTTLLLLD 54 ACEEQNPWARYAELLFPTTLAW 55ACEEQNPWARYAEWLFPTTLLLLE 56 ACEEQNPWARYLEWLFPTETLLLEL 57GGEQNPIYWARYADWLFTTPLLLLDLALLV DADEGT 58 ACEQNPIYWARYADWLFTTPLLLLDLALLV59 WARYADWLFTTPLLLLDLALLV DADEGTCG 60 WARYADWLFTTPLLLLDLALLV DADEGCT 61GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGTCG 62ACEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 63AKEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 64AKEQNPIY WARYADWLFTTPLLLLDLALLV DADECT 65AAEQNPIY WARYADWLFTTALLLLDLALLV DADEGT 66ACAEQNPIY WARYADWLFTTGLLLLDLALLV DADEGT 67AEQNPIY WARYADFLFTTALLLLDLALLV DADE_T 68AEQNPIY FARYADWLFTTPLLLLDLALLV DADEGT 69AEQNPIY FARYADFLFTTPLLLLDLALLW DADE_T 70AKEDQNP_Y WARYADWLFTTPLLLLDLALLV DG_—— 71ACEDQNP_Y WARYADWLFTTPLLLLDLALLV DG_—— 72AEDQNP_Y WARYADWLFTTPLLLLDLALLV DG_—— 73AEDQNP_Y WARYADWLFTTPLLLLELALLV ECG_— 74AKEDQNP_Y WRAYAD_LFT_PLTLLDLLALW DG_—— 75ACEDQNP_Y WRAYAD_LFT_PLTLLDLLALW DG_—— 76AKEDQNDP_Y WARYADWLFTTPLLLLDLALLV G_——— 77TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA 78GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 79 AEQNPIY WARYADWLFTTPL 80AEQNPIY WARYADWLFTTPCL 81 ACEQNPIY WARYADWLFTTPL 82ACEQNPIY FARYADWLFTTPL 83 ACDDQNP WRAYLDLLFPTDTLLLDLLW 84ACEEQNP WRAYLELLFPTETLLLELLW 85 ACDDQNP WARYLDWLFPTDTLLLDL 86CDNNNP WRAYLDLLFPTDTLLLDW 87 ACEEQNP WARYLEWLFPTETLLLEL 88ACEDQNP WARYADWLFPTTLLLLD 89 ACEEQNP WARYAEWLFPTTLLLLE 90ACEDQNP WARYADLLFPTTLAW 91 ACEDQNP WARYAELLFPTTLW 92KEDQNP WARYADLLFPTTLW 93 DDDEDNP IYWARYAHWLFTTPLLLLHGALLVDADECT 94DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 95DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 96DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 97DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 98ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGIG 99ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 100ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 101GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 102GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 103GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG 104AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGTCG 105AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 106GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 107GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 108GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 109AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 110ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG 111DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 112DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 113DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 114DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 115DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 116GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 117AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTCG 118AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 119AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 120GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 121GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 122GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 123GGEQNPIYWARYADWLFTTPLLLLDALLVNANQGT 124DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 125DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 126ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG 127GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 128GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 129GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 130AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 131AAEQNPIYWARYADWLFTDLPLLLLDLLALLVDADEGT 132GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 133GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 134AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT 135AEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 136GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 137GGEQNPIYWAQDYAWLFTTPLLLLDLALLDADEGTCG 138GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 139AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGTCG 140AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG.........EGTK(rhodamine)C(phalloidin)G 141AAEQNPIYWARYADWLFTTPLLLLELALLDADEGTKCG 142AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 143AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (phalloidin)G 144GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 145ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 146ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 147ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 148GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 149DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 150DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 151GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 152AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (phalloidin)G 153AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 154AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 155DDDEDNPIYWARYAHWLFTTPLLLLBGALLVDADECT 156DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 157DDDEDNPIYWARYAHWLFTTPLLLLBGALLVNADECT 158DDDEDNPIYWARYAHWLFTTPLLLLBGALLVNANECT 159DDDEDNPIYWARYADWLFTTPLLLLIBGALLVDADECT 160DDDEDNPIYWARYADWTFTTPLLLLHGALLVDADECT 161DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 162DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 163DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 164DDDEDNPIYWARYHWLFTTPLLLLHGALLVNANECT 165DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 166DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 167DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 168DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 169DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 170GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 171DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 172DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 173DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 174DDDEDNPIYWARYAHMLFTTPLLLLDGALLVDADECT 175DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 176DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 177DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 178DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 179DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 180DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 181AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT 182GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG 183GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 184GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 185GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 186AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 187GGEQNPIYWARYADWLFTTPLLLLDALLVDADEGTCG 188GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 189GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 190GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 191GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 192GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 193GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 194GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 195GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 196GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 197AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 198GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 199GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 200GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 201AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 202AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 203................EGTK(rhidamine)C (phalloidin)G 204AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 205ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 206AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (phalloidin)G 207AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 208AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 209AAEQNPIYWARYADWLFTDLPLLLLDLLALLVDADEGT 210AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 211GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 212GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 213GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 214AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGTCG 215AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 216AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGTCG 217AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTCG 218AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 219AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 220ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG 221ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG 222AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT 223 AEQNPIYFARYADLLFPTTLAW 224AEQNPIYWARYADLLFPTTLAF 225 AEQNPIYWARYADLLFPTTLAW 226ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 227GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 228AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 229AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 230AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT 231 CCTCTTACCTCAGTTACA 232D-Arg8 D-Arg8-CCTCTTACCTCAGTTACA 233 D-Lys4 D-Lys4-CCTCTTACCTCAGTTACA234 S-S-CCTCTTACCTCAGTTACA 235 S-S-CCTCTGACCTCATTTACA 236D-Arg8-Deca D-Arg8-Deca-CCTCTTACCTCAGTTACA 237D-Arg8-Deca-mismatch D-Arg8-Deca- CCTCTGACCTCATTTACA 238S-S-CCTCTTACCTCAGTTACA 239 AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 240AEDQNPYWARYDWLFTTPLLLLDLALLVDCG 241 AEDQNPYWARYADWLFTTPLLLLELALLVECG 242AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT 243ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 244 AE-QN-PI YWARYADWLFTTPLLLLDLALLVDADEGT-COOH 245 AEDQN-P-YWARYADWLFTTPLLLLDLALLV D---G--COOH 246AEDQNDP-YWARYADWLFTTPLLLLDLALLV----G--COOH 247AEQNPI YWARYADFLFTTPLLLLDLALLV DADET-COOH 248AEQNPI YFARYADWLFTTPLLLLDLALLV DADET-COOH 249AEQNPI YFARYADFLFTTPLLLLDLALLW DADET-COOH 250AE-QN-PI YWARYADWLFTTPLLLLDLALLV DADEGCT-COOH 251AEDQN-PI YWARYADWLFTTPLLLLDLALLV DC--G-T-COOH 252AEDQNDPI YWARYADWLFTTPLLLLELALLV EC--G-T-COOH 253Chelate-ACEEQNPWARYLEWLFPTETLLLEL 254AEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT-COOH 255AKEDQNPY WARYADWLFTTPLLLLDLALLV DG-COOH 256AKEDQNDPY WARYADWLFTTPLLLLDLALLV G-COOH 257AEQNPI YWARYADWLFTTPLLLLDLALLV DADEGC-Biotin-T-COOH 258AEDQNP YWARYADWLFTTPLLLLDLALLV DC-Biotin-G-COOH 259AEDQNP YWARYADWLFTTPLLLLELALLV EC-Biotin-G-COOH 260ACEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 261ACEDQNPY WARYADWLFTTPLLLLDLALLV DG 262 ACEDQNPY WRAYADLFTPLTLLDLLALW DG263 ACDDQNP WRAYLDLLFPTDTLLLDLLW 264 WRAYLELLFPTETLLLELLW 265WARYLDWLFPTDTLLLDL 266 WRAYLDLLFPTDTLLLDW 267 WARYLEWLFPTETLLLEL 268WAQYLELLFPTETLLLEW 269 WRAYLELLFPTETLLLEW 270 WARYADWLFPTTLLLLD 271WARYAEWLFPTTLLLLE 272 ACEDQNP WARYADLLFPTTLAW 273ACEEQNP WARYAELLFPTTLAW 274 Ac-TEDAD VLLALDLLLLPTTFLWDAYRAW YPNQECA-Am275 CDDDDDNPNY WARYANWLFTTPLLLLNGALLV EAEET 276CDDDDDNPNY WARYAPWLFTTPLLLLPGALLV EAEET 277Ac-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT 278Ac-AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 279ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGT 280Ac-AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 281DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADET 282CDDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADET 283DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGT 284DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADEGT 285DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANEGT 286AKEDQNDPYWARYADWLFTTPLLLLDLALLVG 287 AEDQNPYWARYADWLFTTPLLLLELALLVCG 288AKDDQNPWRAYLDLLFPTDTLLLDLLWC 289 ACEEQNPWRAYLELLFPTETLLLELLW 290ACDDQNPWARYLDWLFPTDTLLLDL 291 CDNNNPWRAYLDLLFPTDTLLLDW 292CEEQQPWAQYLELLFPTETLLLEW 293 EEQQPWRAYLELLFPTETLLLEW 294CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET 295CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEE 296 AEQNPIYFARYADLLFPTTLAW 297AEQNPIYWARYADLLFPTTLAF 298 AEQNPIYWARYADLLFPTTLAW 299KEDQNPWARYADLLFPTTLW 300 ACEEQNPQAEYAEWLFPTTLLLLE 301AAEEQNPWARYLEWLFPTETLLLEL 302 AKEEQNPWARYLEWLFPTETLLLEL 303AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTGG 304XXEXNPIYWAXXXXXLFTXXLLLXXXALLVXAXXXTXG 305DAAEQNPIYWARYADWLFTTLPLLLLDLLALLVDADEGTKGG 306GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTGG 307XXEXNPIYWAXXXXXLFTXXLLLXXXALLVXAXXXTGG 308DGGEQNDPIYWARYADWLFTTLPLLLLDLLALL VDADEGCTXGG 309AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 310AEDQNPYWARYDWLFTTPLLLLDLALLVDCG 311 GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN

In some embodiments, R⁷ is a peptide having 10 to 50 amino acids. Insome embodiments, R⁷ is a peptide having 20 to 40 amino acids. In someembodiments, R⁷ is a peptide having 20 to 40 amino acids. In someembodiments, R⁷ is a peptide having 10 to 20 amino acids. In someembodiments, R⁷ is a peptide having 20 to 30 amino acids. In someembodiments, R⁷ is a peptide having 30 to 40 amino acids.

Suitable therapeutic molecules (e.g., R⁸) for use in the inventioninclude those which have undesirable side effects when deliveredsystemically because of their possible deleterious effect on normaltissue. Example therapeutic molecules are DNA repair inhibitingcompounds such as inhibitors of PARP, ATR, DNK-PK, and ATM. Such DNArepair inhibiting compound are useful for treatment of cancer and otherdiseases in which inhibition of DNA repair would be desirable.

Three PARP inhibitors (olaparib, rucaparib, and niraparib) are currentlycommercially available and others are in development, such as AG-014699(Agouron/Pfizer), KU-0059436 (KuDOS/AstraZeneca), INO-1001(Inotek/Genentech), NT-125 (now E-7449; Eisai;3H-Pyridazino[3,4,5-de]quinazolin-3-one,8-[(1,3-dihydro-2H-isoindol-2-yl)methyl]-1,2-dihydro-), 2×-121 (2×Oncology; 3H-pyridazino[3,4,5-de]quinazolin-3-one,8-[(1,3-dihydro-2H-isoindol-2-yl)methyl]-1,2-dihydro-), and ABT-888(Abbvie). PARP inhibitors are disclosed in (for example) U.S. Pat. Nos.6,100,283; 6,310,082; 6,495,541; 6,548,494; 6,696,437; 7,151,102;7,196,085; 7,449,464; 7,692,006; 7,781,596; 8,067,613; 8,071,623; and8,697,736, which patents are incorporated herein by reference in theirentirety.

In some embodiments, R¹ and R² are each independently selected from Hand methyl, and R³, R⁴, R⁵, and R⁶ are each hydrogen.

In some embodiments, R¹ and R² are each independently selected from Hand methyl.

In some embodiments, R¹ and R² are each H.

In some embodiments, R³ and R⁴ are each H.

In some embodiments, R⁵ and R⁶ are each H.

In some embodiments, R⁹, R¹⁰, R¹¹, and R¹² are each independentlyselected from H and methyl.

In some embodiments, the enzyme capable of cleaving [AA]x is CathepsinB, matrix-metalloproteinases (MMP), DPPIV, glycoprotein, peptidase, orcaspase. In some embodiments, [AA]x is a peptide having two to twelve (xis 2 to 12) amino acid (AA) residues. In some embodiments, [AA]x is apeptide having two to ten (x is 2 to 10) amino acid (AA) residues. Insome embodiments, [AA]x is a peptide having two to five (x is 2 to 5)amino acid (AA) residues. In some embodiments, [AA]x is a peptide havingsix to nine (x is 6 to 9) amino acid (AA) residues. In some embodiments,[AA]x is a peptide having three to eight (x is 3 to 8) amino acid (AA)residues.

Peptide linkers (e.g., [AA]x) that are capable of being cleaved byproteins are described in Yang, Y., Acta Pharmaceutica Sinica B 2011,1(3), 143-159; Choi, K., Theranostics 2012, 2, 156-178; and Anderson,C., Ind. Eng. Chem. Res. 2017, 56, 4761-5777.

Peptide linkers (e.g., [AA]x) that are capable of being cleaved by DPPIVare described in Diez-Torrubia, A., J. Med. Chem. 2010, 53, 559-572;Garcia-Aparicio, C., J. Med. Chem. 2006, 49, 5339-5351; Diez-Torrubia,A., ChemMedChem 2012, 7, 618-628; Dahan, A., Mol. Pharmaceutics 2014,11, 4385-4394; Wickstrom, M., Oncotarget 2017, 8, 66641-66655; andSimplicio, A. L., Molecules, 2008, 13, 519-547.

Peptide linkers (e.g., [AA]x) that are capable of bring cleaved bycathepsin B are described in Caculitan, N., Cancer Res. 2017, 77(24),7027-7037; Zhong, Y-J, International Journal of Oncology 2013, 42,373-383; and Fan, P., Drug Metabolism and Disposition 2016, 44,1253-1261.

Peptide linkers (e.g., [AA]x) that are capable of bring cleaved by MMP(e.g., MMP-9) are described in Kalafatovic, D., Biomaterials 98 (2016),192-202; Kim, H S, Gene Therapy 2013, 20, 378-385; and Yao, W., Trendsin Pharmacological Sciences 2018, 39, 766-781.

In some embodiments, [AA]x is -Pro_Gly-; -Val_Cit-,-Gly_Pro_Leu_Gly_Leu_Ala_Gly_Asp_Asp-, -Gly_Pro_GLeu_Gly_Val_Arg_Gly, or-Ser_Ser_Lys_Leu_Gly-.

In some embodiments, S1 is a group having the following structure:

In some embodiments, S1 is a group having the following structure:

The S1 group can be a carbohydrate that can be cleaved by glucuronidase.Such carbohydrate groups are used in the art as glucuronide prodrugs,and are described in Grinda, M. Med. Chem. Commun. 2012, 3, 68-70;Herceg, V., Biorganic Chemistry 2018, 78, 372-380; Adiyala P.,Bioorganic Chemistry 2018, 76, 288-293; and Kolakowski, R., Angew. Chem.Int. Ed. 2016, 55, 7948-7951.

Also provided herein is a compound of formula (I)R8-Q-R7   (I)

-   -   and the pharmaceutically acceptable salts thereof, wherein    -   R⁸ is a member selected from the group consisting of:

-   -   Q is a member selected from the group consisting of

wherein R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from H,optionally substituted C₁-C₄ alkyl, optionally substituted C₁-C₄alkenyl, optionally substituted C₁-C₄ alkoxy, amino optionallysubstituted with one or two C₁-C₄ alkyl substituents, halo, nitro orhydroxyl, and wherein [NH,O,S} means a linking NH, O, or S moiety,

and

R⁷ is a peptide capable of selectively delivering R⁸Q across a cellmembrane having an acidic or hypoxic mantle in a pH dependent fashion.

In some embodiments, R⁷ is a member selected from the group consistingof

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG, (SEQ ID NO. 2; Pv2)AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, and (SEQ ID NO. 3; Pv3)ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG,

wherein R⁷ and Q are attached through a cysteine residue.

In some embodiments, R¹ and R² are each independently selected from Hand methyl and R³, R⁴, R⁵, and R⁶ are each hydrogen.

In some embodiments, R⁷ is ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1;Pv1).

In some embodiments, R⁷ is AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG (SEQ IDNO. 2; Pv2).

In some embodiments, R⁷ is ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG (SEQ ID NO.3; Pv3).

In some embodiments, the compound of formula (I) is selected from:

The molecules of the invention can be tagged, for example, with a probesuch as a fluorophore, radioisotope, and the like. In some embodiments,the probe is a fluorescent probe, such as LICOR. A fluorescent probe caninclude any moiety that can re-emit light upon light excitation (e.g., afluorophore).

The Amino acids are represented by the IUPAC abbreviations, as follows:Alanine (Ala; A), Arginine (Arg; R), Asparagine (Asn; N), Aspartic acid(Asp; D), Cysteine (Cys; C), Glutamine (Gln; Q), Glutamic acid (Glu; E),Glycine (Gly; G), Histidine (His; H), Isoleucine (Ile; I), Leucine (Leu;L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F),Proline (Pro; P), Serine (Ser; S), Threonine (Thr; T), Tryptophan (Trp;W), Tyrosine (Tyr; Y), Valine (Val; V). The term “Pv1” meansADDQNPWRAYLDLLFPTDTLLLDLLWCG, which is the peptide of SEQ ID No. 1. Theterm “Pv2” means AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, which is thepeptide of SEQ ID No. 2. The term “Pv3” meansADDQNPWRAYLDLLFPTDTLLLDLLWDADECG, which is the peptide of SEQ ID No. 3.The term “Pv4” means Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG, whichis the peptide of SEQ ID NO. 4. The term “Pv5” meansAAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC, which is the peptide of SEQ IDNO. 5. In the compounds of the invention, the peptides R⁷ are attachedto the disulfide linker by the cysteine moiety.

The term “acidic and/or hypoxic mantle” refers to the environment of thecell in the diseased tissue in question having a pH lower than 7.0 andpreferably lower than 6.5. An acidic or hypoxic mantle more preferablyhas a pH of about 5.5 and most preferably has a pH of about 5.0. Thecompounds of formula (I) insert across a cell membrane having an acidicand/or hypoxic mantle in a pH dependent fashion to insert R⁸Q into thecell, whereupon the disulfide linker is cleaved to deliver free R⁸H.Since the compounds of formula (I) are pH-dependent, they preferentiallyinsert across a cell membrane only in the presence of an acidic orhypoxic mantle surrounding the cell and not across the cell membrane of“normal” cells, which do not have an acidic or hypoxic mantle.

The terms “pH-sensitive” or “pH-dependent” as used herein to refer tothe peptide R₇ or to the mode of insertion of the peptide R⁷ or of thecompounds of the invention across a cell membrane, means that thepeptide has a higher affinity to a cell membrane lipid bilayer having anacidic or hypoxic mantle than a membrane lipid bilayer at neutral pH.Thus, the compounds of the invention preferentially insert through thecell membrane to insert R⁸Q to the interior of the cell (and thusdeliver R⁸H as described above) when the cell membrane lipid bilayer hasan acidic or hypoxic mantle (a “diseased” cell) but does not insertthrough a cell membrane when the mantle (the environment of the cellmembrane lipid bilayer) is not acidic or hypoxic (a “normal” cell). Itis believed that this preferential insertion is achieved as a result ofthe peptide R⁷ forming a helical configuration, which facilitatesmembrane insertion.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment (while theembodiments are intended to be combined as if written in multiplydependent form). Conversely, various features of the invention whichare, for brevity, described in the context of a single embodiment, canalso be provided separately or in any suitable subcombination. Thus, itis contemplated as features described as embodiments of the compounds ofFormula (I) can be combined in any suitable combination.

At various places in the present specification, certain features of thecompounds are disclosed in groups or in ranges. It is specificallyintended that such a disclosure include each and every individualsubcombination of the members of such groups and ranges. For example,the term “C₁₋₆ alkyl” is specifically intended to individually disclose(without limitation) methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl and C₆alkyl.

The term “n-membered,” where n is an integer, typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, piperidinyl is an example of a6-membered heterocycloalkyl ring, pyrazolyl is an example of a5-membered heteroaryl ring, pyridyl is an example of a 6-memberedheteroaryl ring and 1,2,3,4-tetrahydro-naphthalene is an example of a10-membered cycloalkyl group.

At various places in the present specification, variables definingdivalent linking groups may be described. It is specifically intendedthat each linking substituent include both the forward and backwardforms of the linking substituent. For example, —NR(CR′R″)_(n)— includesboth —NR(CR′R″)_(n)— and —(CR′R″)_(n)NR— and is intended to discloseeach of the forms individually. Where the structure requires a linkinggroup, the Markush variables listed for that group are understood to belinking groups. For example, if the structure requires a linking groupand the Markush group definition for that variable lists “alkyl” or“aryl” then it is understood that the “alkyl” or “aryl” represents alinking alkylene group or arylene group, respectively.

The term “substituted” means that an atom or group of atoms formallyreplaces hydrogen as a “substituent” attached to another group. The term“substituted”, unless otherwise indicated, refers to any level ofsubstitution, e.g., mono-, di-, tri-, tetra- or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.It is to be understood that substitution at a given atom is limited byvalency. It is to be understood that substitution at a given atomresults in a chemically stable molecule. The phrase “optionallysubstituted” means unsubstituted or substituted. The term “substituted”means that a hydrogen atom is removed and replaced by a substituent. Asingle divalent substituent, e.g., oxo, can replace two hydrogen atoms.

The term “C_(n-m)” indicates a range which includes the endpoints,wherein n and m are integers and indicate the number of carbons.Examples include C₁₋₄, C₁₋₆ and the like.

The term “alkyl” employed alone or in combination with other terms,refers to a saturated hydrocarbon group that may be straight-chained orbranched. The term “C_(n-m) alkyl”, refers to an alkyl group having n tom carbon atoms. An alkyl group formally corresponds to an alkane withone C—H bond replaced by the point of attachment of the alkyl group tothe remainder of the compound. In some embodiments, the alkyl groupcontains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moietiesinclude, but are not limited to, chemical groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higherhomologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl,1,2,2-trimethylpropyl and the like.

The term “alkenyl” employed alone or in combination with other terms,refers to a straight-chain or branched hydrocarbon group correspondingto an alkyl group having one or more double carbon-carbon bonds. Analkenyl group formally corresponds to an alkene with one C—H bondreplaced by the point of attachment of the alkenyl group to theremainder of the compound. The term “C_(n-m) alkenyl” refers to analkenyl group having n to m carbons. In some embodiments, the alkenylmoiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Example alkenylgroups include, but are not limited to, ethenyl, n-propenyl,isopropenyl, n-butenyl, sec-butenyl and the like.

The term “alkynyl” employed alone or in combination with other terms,refers to a straight-chain or branched hydrocarbon group correspondingto an alkyl group having one or more triple carbon-carbon bonds. Analkynyl group formally corresponds to an alkyne with one C—H bondreplaced by the point of attachment of the alkyl group to the remainderof the compound. The term “C_(n-m) alkynyl” refers to an alkynyl grouphaving n to m carbons. Example alkynyl groups include, but are notlimited to, ethynyl, propyn-1-yl, propyn-2-yl and the like. In someembodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3carbon atoms.

The term “alkylene”, employed alone or in combination with other terms,refers to a divalent alkyl linking group. An alkylene group formallycorresponds to an alkane with two C—H bond replaced by points ofattachment of the alkylene group to the remainder of the compound. Theterm “C_(n-m) alkylene” refers to an alkylene group having n to m carbonatoms. Examples of alkylene groups include, but are not limited to,ethan-1,2-diyl, ethan-1,1-diyl, propan-1,3-diyl, propan-1,2-diyl,propan-1,1-diyl, butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl,2-methyl-propan-1,3-diyl and the like.

The term “amino” refers to a group of formula —NH₂.

The term “carbonyl”, employed alone or in combination with other terms,refers to a —C(═O)— group, which also may be written as C(O).

The term “cyano” or “nitrile” refers to a group of formula —CN, whichalso may be written as —CN.

The terms “halo” or “halogen”, used alone or in combination with otherterms, refers to fluoro, chloro, bromo and iodo. In some embodiments,“halo” refers to a halogen atom selected from F, Cl, or Br. In someembodiments, halo groups are F.

The term “haloalkyl” as used herein refers to an alkyl group in whichone or more of the hydrogen atoms has been replaced by a halogen atom.The term “C_(n-m) haloalkyl” refers to a C_(n-m) alkyl group having n tom carbon atoms and from at least one up to {2(n to m)+1} halogen atoms,which may either be the same or different. In some embodiments, thehalogen atoms are fluoro atoms. In some embodiments, the haloalkyl grouphas 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF₃,C₂F₅, CHF₂, CH₂F, CCl₃, CHCl₂, C₂Cl₅ and the like. In some embodiments,the haloalkyl group is a fluoroalkyl group.

The term “haloalkoxy”, employed alone or in combination with otherterms, refers to a group of formula —O-haloalkyl, wherein the haloalkylgroup is as defined above. The term “C_(n-m) haloalkoxy” refers to ahaloalkoxy group, the haloalkyl group of which has n to m carbons.Example haloalkoxy groups include trifluoromethoxy and the like. In someembodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbonatoms.

The term “oxo” refers to an oxygen atom as a divalent substituent,forming a carbonyl group when attached to carbon, or attached to aheteroatom forming a sulfoxide or sulfone group, or an N-oxide group. Insome embodiments, heterocyclic groups may be optionally substituted by 1or 2 oxo (═O) substituents.

The term “oxidized” in reference to a ring-forming N atom refers to aring-forming N-oxide.

The term “oxidized” in reference to a ring-forming S atom refers to aring-forming sulfonyl or ring-forming sulfinyl.

The term “aromatic” refers to a carbocycle or heterocycle having one ormore polyunsaturated rings having aromatic character (i.e., having(4n+2) delocalized π (pi) electrons where n is an integer).

The term “aryl,” employed alone or in combination with other terms,refers to an aromatic hydrocarbon group, which may be monocyclic orpolycyclic (e.g., having 2 fused rings). The term “C_(n-m) aryl” refersto an aryl group having from n tom ring carbon atoms. Aryl groupsinclude, e.g., phenyl, naphthyl, and the like. In some embodiments, arylgroups have from 6 to about 10 carbon atoms. In some embodiments arylgroups have 6 carbon atoms. In some embodiments aryl groups have 10carbon atoms. In some embodiments, the aryl group is phenyl.

The term “heteroaryl” or “heteroaromatic,” employed alone or incombination with other terms, refers to a monocyclic or polycyclicaromatic heterocycle having at least one heteroatom ring member selectedfrom sulfur, oxygen and nitrogen. In some embodiments, the heteroarylring has 1, 2, 3 or 4 heteroatom ring members independently selectedfrom nitrogen, sulfur and oxygen. In some embodiments, any ring-formingN in a heteroaryl moiety can be an N-oxide. In some embodiments, theheteroaryl has 5-14 ring atoms including carbon atoms and 1, 2, 3 or 4heteroatom ring members independently selected from nitrogen, sulfur andoxygen. In some embodiments, the heteroaryl has 5-10 ring atomsincluding carbon atoms and 1, 2, 3 or 4 heteroatom ring membersindependently selected from nitrogen, sulfur and oxygen. In someembodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatomring members independently selected from nitrogen, sulfur and oxygen. Insome embodiments, the heteroaryl is a five-membered or six-memberedheteroaryl ring. In other embodiments, the heteroaryl is aneight-membered, nine-membered or ten-membered fused bicyclic heteroarylring.

A five-membered heteroaryl ring is a heteroaryl group having five ringatoms wherein one or more (e.g., 1, 2 or 3) ring atoms are independentlyselected from N, O and S.

A six-membered heteroaryl ring is a heteroaryl group having six ringatoms wherein one or more (e.g., 1, 2 or 3) ring atoms are independentlyselected from N, O and S.

The term “cycloalkyl,” employed alone or in combination with otherterms, refers to a non-aromatic hydrocarbon ring system (monocyclic,bicyclic or polycyclic), including cyclized alkyl and alkenyl groups.The term “C_(n-m) cycloalkyl” refers to a cycloalkyl that has n to mring member carbon atoms. Cycloalkyl groups can include mono- orpolycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles.Cycloalkyl groups can have 3, 4, 5, 6 or 7 ring-forming carbons (C₃₋₇).In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to5 ring members, or 3 to 4 ring members. In some embodiments, thecycloalkyl group is monocyclic. In some embodiments, the cycloalkylgroup is monocyclic or bicyclic. In some embodiments, the cycloalkylgroup is a C₃₋₆ monocyclic cycloalkyl group. Ring-forming carbon atomsof a cycloalkyl group can be optionally oxidized to form an oxo orsulfido group. Cycloalkyl groups also include cycloalkylidenes. In someembodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl. Also included in the definition of cycloalkyl are moietiesthat have one or more aromatic rings fused (i.e., having a bond incommon with) to the cycloalkyl ring, e.g., benzo or thienyl derivativesof cyclopentane, cyclohexane and the like. A cycloalkyl group containinga fused aromatic ring can be attached through any ring-forming atomincluding a ring-forming atom of the fused aromatic ring. Examples ofcycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl,and the like. In some embodiments, the cycloalkyl group is cyclopropyl,cyclobutyl, cyclopentyl, or cyclohexyl.

The term “heterocycloalkyl,” employed alone or in combination with otherterms, refers to a non-aromatic ring or ring system, which mayoptionally contain one or more alkenylene groups as part of the ringstructure, which has at least one heteroatom ring member independentlyselected from nitrogen, sulfur, oxygen and phosphorus, and which has4-10 ring members, 4-7 ring members, or 4-6 ring members. Includedwithin the term “heterocycloalkyl” are monocyclic 4-, 5-, 6- and7-membered heterocycloalkyl groups. Heterocycloalkyl groups can includemono- or bicyclic (e.g., having two fused or bridged rings) orspirocyclic ring systems. In some embodiments, the heterocycloalkylgroup is a monocyclic group having 1, 2 or 3 heteroatoms independentlyselected from nitrogen, sulfur and oxygen. Ring-forming carbon atoms andheteroatoms of a heterocycloalkyl group can be optionally oxidized toform an oxo or sulfido group or other oxidized linkage (e.g., C(O),S(O), C(S) or S(O)₂, N-oxide etc.) or a nitrogen atom can bequaternized. The heterocycloalkyl group can be attached through aring-forming carbon atom or a ring-forming heteroatom. In someembodiments, the heterocycloalkyl group contains 0 to 3 double bonds. Insome embodiments, the heterocycloalkyl group contains 0 to 2 doublebonds. Also included in the definition of heterocycloalkyl are moietiesthat have one or more aromatic rings fused (i.e., having a bond incommon with) to the heterocycloalkyl ring, e.g., benzo or thienylderivatives of piperidine, morpholine, azepine, etc. A heterocycloalkylgroup containing a fused aromatic ring can be attached through anyring-forming atom including a ring-forming atom of the fused aromaticring. Examples of heterocycloalkyl groups include 2pyrrolidinyl;morpholinul; azetidinyl; and piperazinyl.

At certain places, the definitions or embodiments refer to specificrings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwiseindicated, these rings can be attached to any ring member provided thatthe valency of the atom is not exceeded. For example, an azetidine ringmay be attached at any position of the ring, whereas an azetidin-3-ylring is attached at the 3-position.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent invention that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically inactive startingmaterials are known in the art, such as by resolution of racemicmixtures or by stereoselective synthesis. Many geometric isomers ofolefins, C═N double bonds and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. One method includes fractionalrecrystallization using a chiral resolving acid which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, e.g., optically active acids,such as the D and L forms of tartaric acid, diacetyltartaric acid,dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or thevarious optically active camphorsulfonic acids such as β-camphorsulfonicacid. Other resolving agents suitable for fractional crystallizationmethods include stereoisomerically pure forms of α-methylbenzylamine(e.g., S and R forms, or diastereomerically pure forms),2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

In some embodiments, the compounds of the invention have the(R)-configuration. In other embodiments, the compounds have the(S)-configuration. In compounds with more than one chiral centers, eachof the chiral centers in the compound may be independently (R) or (S),unless otherwise indicated.

Compounds of the invention also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone-enol pairs, amide-imidic acidpairs, lactam-lactim pairs, enamine-imine pairs, and annular forms wherea proton can occupy two or more positions of a heterocyclic system,e.g., 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can be inequilibrium or sterically locked into one form by appropriatesubstitution.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium. One ormore constituent atoms of the compounds of the invention can be replacedor substituted with isotopes of the atoms in natural or non-naturalabundance. In some embodiments, the compound includes at least onedeuterium atom. For example, one or more hydrogen atoms in a compound ofthe present disclosure can be replaced or substituted by deuterium. Insome embodiments, the compound includes two or more deuterium atoms. Insome embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12 deuterium atoms. Synthetic methods for including isotopes intoorganic compounds are known in the art (Deuterium Labeling in OrganicChemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts,1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau,Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007,7744-7765; The Organic Chemistry of Isotopic Labelling by James R.Hanson, Royal Society of Chemistry, 2011). Isotopically labeledcompounds can used in various studies such as NMR spectroscopy,metabolism experiments, and/or assays.

Substitution with heavier isotopes such as deuterium, may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample, increased in vivo half-life or reduced dosage requirements, andhence may be preferred in some circumstances. (A. Kerekes et. al. J.Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm.2015, 58, 308-312).

The term, “compound,” as used herein is meant to include allstereoisomers, geometric isomers, tautomers and isotopes of thestructures depicted. The term is also meant to refer to compounds of theinventions, regardless of how they are prepared, e.g., synthetically,through biological process (e.g., metabolism or enzyme conversion), or acombination thereof.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.,hydrates and solvates) or can be isolated. When in the solid state, thecompounds described herein and salts thereof may occur in various formsand may, e.g., take the form of solvates, including hydrates. Thecompounds may be in any solid state form, such as a polymorph orsolvate, so unless clearly indicated otherwise, reference in thespecification to compounds and salts thereof should be understood asencompassing any solid state form of the compound.

In some embodiments, the compounds of the invention, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, e.g., a composition enriched in the compounds of the invention.Substantial separation can include compositions containing at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, or at leastabout 99% by weight of the compounds of the invention, or salt thereof.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions, “ambient temperature” and “room temperature,” as usedherein, are understood in the art, and refer generally to a temperature,e.g., a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, e.g., a temperature from about 20°C. to about 30° C.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. The term “pharmaceutically acceptablesalts” refers to derivatives of the disclosed compounds wherein theparent compound is modified by converting an existing acid or basemoiety to its salt form. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. The pharmaceutically acceptable saltsof the present invention include the non-toxic salts of the parentcompound formed, e.g., from non-toxic inorganic or organic acids. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, alcohols (e.g., methanol, ethanol,iso-propanol or butanol) or acetonitrile (MeCN) are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J.Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook ofPharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). Insome embodiments, the compounds described herein include the N-oxideforms.

As understood in the art, the symbol

is used in structural formulas to indicate the bond that is the point ofattachment of the moiety containing it to the adjacent portion of thecompound. According to a similar convention, pendant carbon atoms andtheir attached hydrogen atoms may not be explicitly expressed. Thus, thesymbol

represents a methyl group, the symbol

represents an ethyl group, the symbol

represents a cyclopentyl group, and so on. The orientation of asubstituent group in a molecule or moiety is represented by theconvention that the symbol

represents a bond in which the group is projecting out of the plane ofthe page toward the reader, while the symbol

represents a bond in which the group is projecting behind the plane ofthe page away from the reader, and the symbol

represents a bond in which the group has indeterminate orientation(i.e., the compound is diasteriomeric).

All terms as used herein in this specification, unless otherwise stated,shall be understood in their ordinary meaning as known in the art. Forexample, “C₁₋₄alkyl” is a saturated aliphatic hydrocarbon monovalentradical containing 1-4 carbons such as methyl, ethyl, n-propyl,1-methylethyl (isopropyl), n-butyl or t-butyl; “C₁₋₄ alkoxy” is a C₁₋₄alkyl with a terminal oxygen, such as methoxy, ethoxy, propoxy, butoxy.All alkyl, alkenyl and alkynyl groups shall be understood as beingbranched or unbranched, cyclized or uncyclized where structurallypossible and unless otherwise specified. Other more specific definitionsare as follows:

The term “C_(1-n)-alkyl”, wherein n is an integer from 2 to 6, eitheralone or in combination with another radical denotes an acyclic,saturated, branched or linear hydrocarbon radical with 1 to n C atoms.For example the term C₁₋₅-alkyl embraces the radicals H₃C—, H₃C—CH₂—,H₃C—CH₂—CH₂—, H₃C—CH(CH₃)—, H₃C—CH₂—CH₂—CH₂—, H₃C—CH₂—CH(CH₃)—,H₃C—CH(CH₃)—CH₂—, H₃C—C(CH₃)₂—, H₃C—CH₂—CH₂—CH₂—CH₂—,H₃C—CH₂—CH₂—CH(CH₃)—, H₃C—CH₂—CH(CH₃)—CH₂—, H₃C—CH(CH₃)—CH₂—CH₂—,H₃C—CH₂—C(CH₃)₂—, H₃C—C(CH₃)₂—CH₂—, H₃C—CH(CH₃)—CH(CH₃)— andH₃C—CH₂—CH(CH₂CH₃)—.

The term “C_(3-n)-cycloalkyl”, wherein n is an integer 4 to 7, eitheralone or in combination with another radical denotes a cyclic,saturated, unbranched hydrocarbon radical with 3 to n C atoms. Forexample, the term C₃₋₇-cycloalkyl includes cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and cycloheptyl.

The term “heteroatom” as used herein shall be understood to mean atomsother than carbon such as O, N, S and P.

In some embodiments, alkyl groups or carbon chains, one or more carbonatoms can be optionally replaced by heteroatoms: O, S or N. It shall beunderstood that if N is not represented as substituted then it is NH,and that the heteroatoms may replace either terminal carbon atoms orinternal carbon atoms within a branched or unbranched carbon chain. Suchgroups can be substituted as herein above described by groups such asoxo to result in definitions such as but not limited to: alkoxycarbonyl,acyl, amido and thioxo.

In some embodiments, the term “aryl” as used herein, either alone or incombination with another radical, denotes a carbocyclic aromaticmonocyclic group containing 6 carbon atoms which may be further fused toa second 5- or 6-membered carbocyclic group which may be aromatic,saturated or unsaturated. Aryl includes, but is not limited to, phenyl,indanyl, indenyl, naphthyl, anthracenyl, phenanthrenyl,tetrahydronaphthyl and dihydronaphthyl.

In some embodiments, the term “heteroaryl” (sometimes abbreviated“hetaryl”) means an aromatic 5 to 6-membered monocyclic heteroaryl or anaromatic 7 to 11-membered heteroaryl bicyclic ring where at least one ofthe rings is aromatic, wherein the heteroaryl ring contains 1-4heteroatoms such as N, O and S. Non-limiting examples of 5 to 6-memberedmonocyclic heteroaryl rings include furanyl, oxazolyl, isoxazolyl,oxadiazolyl, thiazolyl, pyrazolyl, pyrrolyl, imidazolyl, tetrazolyl,triazolyl, thienyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyridazinyl,pyrazinyl, triazinyl, and purinyl. Non-limiting examples of 7 to11-membered heteroaryl bicyclic heteroaryl rings include benzimidazolyl,quinolinyl, dihydro-2H-quinolinyl, tetrahydroquinolinyl, isoquinolinyl,quinazolinyl, indazolyl, thieno[2,3-d]pyrimidinyl, indolyl, isoindolyl,benzofuranyl, dihydrobenzofuranyl, benzopyranyl, benzodioxolyl,benzoxazolyl and benzothiazolyl.

As used in Q, the definition of “aryl and hetaryl” are as describedabove. They are preferably phenyl or imidazoyl such as the followingmoieties that in conjunction with [NH,O,S] chemically undergo immolationto release R₈H as described below.

In some embodiments, the term “heterocyclyl” means a stable non-aromatic4-8 membered monocyclic heterocyclic radical or a stable nonaromatic 6to 11-membered fused bicyclic, bridged bicyclic or spirocyclicheterocyclic radical. The 5 to 11-membered heterocycle consists ofcarbon atoms and one or more, preferably from one to four heteroatomschosen from nitrogen, oxygen and sulfur. The heterocycle may be eithersaturated or partially unsaturated. Non-limiting examples of nonaromatic4-8 membered monocyclic heterocyclic radicals include tetrahydrofuranyl,azetidinyl, pyrrolidinyl, pyranyl, tetrahydropyranyl, dioxanyl,thiomorpholinyl, 1,1-dioxo-1λ⁶-thiomorpholinyl, morpholinyl,piperidinyl, piperazinyl, and azepinyl. Non-limiting examples ofnonaromatic 6 to 11-membered fused bicyclic radicals includeoctahydroindolyl, octahydrobenzofuranyl, and octahydrobenzothiophenyl.Non-limiting examples of nonaromatic 6 to 11-membered bridged bicyclicradicals include 2-azabicyclo[2.2.1]heptanyl,3-azabicyclo[3.1.0]hexanyl, and 3-azabicyclo[3.2.1]octanyl. Non-limitingexamples of nonaromatic 6 to 11-membered spirocyclic heterocyclicradicals include 7-aza-spiro[3,3]heptanyl, 7-spiro[3,4]octanyl, and7-aza-spiro[3,4]octanyl. The term “heterocyclyl” or is intended toinclude all the possible isomeric forms.

The term “halogen” and the corresponding term “halo” as used in thepresent specification shall be understood to mean bromine, chlorine,fluorine or iodine, or the corresponding bromo, chloro, fluoro, or iodo.The definitions “halogenated”, “partially or fully halogenated”;partially or fully fluorinated; “substituted by one or more halogenatoms”, includes for example, mono, di or tri halo derivatives on one ormore carbon atoms. For alkyl, non-limiting examples would be —CH₂CHF₂,—CF₃, etc.

Each alkyl, cycloalkyl, heterocycle, aryl or heteroaryl, or the analogsthereof, described herein shall be understood to be optionally partiallyor fully halogenated.

As used herein, “nitrogen” or “N” and “sulfur” or “S” includes anyoxidized form of nitrogen and sulfur and the quaternized form of anybasic nitrogen. For example, for an —S—C₁₋₆ alkyl radical, unlessotherwise specified, this shall be understood to include —S(O)—C₁₋₆alkyl and —S(O)₂—C₁₋₆ alkyl, likewise, —S—R_(a) may be represented asphenyl-S(O)_(m)— when R_(a) is phenyl and where m is 0, 1 or 2.

Unless specifically indicated, throughout the specification and theappended claims, a given chemical formula or name shall encompasstautomers and all stereo, optical and geometrical isomers (e.g.enantiomers, diastereomers, E/Z isomers, etc.) and racemates thereof aswell as mixtures in different proportions of the separate enantiomers,mixtures of diastereomers, or mixtures of any of the foregoing formswhere such isomers and enantiomers exist, as well as salts, includingpharmaceutically acceptable salts thereof and solvates thereof such asfor instance hydrates including solvates of the free compounds orsolvates of a salt of the compound. In the peptides R₇, the amino acidsmay be all of L configuration, all of D configuration, or a mixture of Dand L configuration.

Compounds of the invention also include their isotopically-labelledforms. An isotopically-labelled form of a compound of the presentinvention is identical to said active agent but for the fact that one ormore atoms of said compound have been replaced by an atom or atomshaving an atomic mass or mass number different from the atomic mass ormass number of said atom which is usually found in nature. Examples ofisotopes which are readily available commercially and which can beincorporated into a compound of the present invention in accordance withwell established procedures, include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, fluorine and chlorine, e.g., ²H(deuterium or “D”), ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and³⁶Cl, respectively. A compound of the present invention, a prodrugthereof, or a pharmaceutically acceptable salt of either which containsone or more of the above-mentioned isotopes and/or other isotopes ofother atoms is contemplated to be within the scope of the presentinvention.

The invention includes pharmaceutically acceptable derivatives ofcompounds of formula (I). The term “pharmaceutically acceptablederivative” refers to any pharmaceutically acceptable salt or ester, orany other compound which, upon administration to a patient, is capableof providing (directly or indirectly) a compound useful for theinvention, or a pharmacologically active metabolite or pharmacologicallyactive residue thereof. A pharmacologically active metabolite shall beunderstood to mean any compound of the invention capable of beingmetabolized enzymatically or chemically. This includes, for example,hydroxylated or oxidized derivative compounds of the formula (I).

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. For example,such salts include acetates, ascorbates, benzenesulfonates, benzoates,besylates, bicarbonates, bitartrates, bromides/hydrobromides, edetates,camsylates, carbonates, chlorides/hydrochlorides, citrates, edisylates,ethane disulfonates, estolates esylates, fumarates, gluceptates,gluconates, glutamates, glycolates, glycollylarsnilates,hexylresorcinates, hydrabamines, hydroxymaleates, hydroxynaphthoates,iodides, isothionates, lactates, lactobionates, malates, maleates,mandelates, methanesulfonates, methylbromides, methylnitrates, methylsulfates, mucates, napsylates, nitrates, oxalates, pamoates,pantothenates, phenylacetates, phosphates/diphosphates,polygalacturonates, propionates, salicylates, stearates, subacetates,succinates, sulfamides, sulfates, tannates, tartrates, teoclates,toluenesulfonates, triethiodides, ammonium, benzathines,chloroprocaines, cholines, diethanolamines, ethylenediamines, megluminesand procaines. Further pharmaceutically acceptable salts can be formedwith cations from metals like aluminium, calcium, lithium, magnesium,potassium, sodium, zinc and the like. (See, e.g., Pharmaceutical Salts,Birge, S. M. et al., J. Pharm. Sci., (1977), 66, 1-19).

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha sufficient amount of the appropriate base or acid in water or in anorganic diluent like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile, or a mixture thereof.

Salts of other acids than those mentioned above which for example areuseful for purifying or isolating the compounds of the present invention(e.g. trifluoro acetate salts) also comprise a part of the invention.

In addition, within the scope of the invention are prodrugs of thecompounds of the formula (I). Prodrugs include those compounds that,upon simple chemical transformation, are modified to produce compoundsof the invention. Simple chemical transformations include hydrolysis,oxidation and reduction. Specifically, when a prodrug is administered toa patient, the prodrug may be transformed into a compound disclosedhereinabove, thereby imparting the desired pharmacological effect. Byreserving the right to proviso out or exclude any individual members ofany such group, including any sub-ranges or combinations of sub-rangeswithin the group, that can be claimed according to a range or in anysimilar manner, less than the full measure of this disclosure can beclaimed for any reason. Further, by reserving the right to proviso outor exclude any individual substituents, analogs, compounds, ligands,structures, or groups thereof, or any members of a claimed group, lessthan the full measure of this disclosure can be claimed for any reason.

The terms “a,” “an,” and “the” are intended to include pluralalternatives, e.g., at least one, unless otherwise specified. Forinstance, the disclosure of “a therapeutic agent” or “a compound” ismeant to encompass one, or mixtures or combinations of more than one,therapeutic agent or compound, respectively.

While compositions and methods are described in terms of “comprising”various components or steps, the compositions and methods can also“consist essentially of” or “consist of” the various components orsteps. For example, a pharmaceutical composition described herein cancomprise; alternatively, can consist essentially of; or alternatively,can consist of; (i) a therapeutically effective amount of a compound, ora pharmaceutically acceptable salt thereof, and (ii) a pharmaceuticallyacceptable diluent, excipient, or carrier.

Synthesis

Compounds of the invention, including salts thereof, can be preparedusing known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes, such as those inthe Schemes below.

The reactions for preparing compounds of the invention can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediatesor products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups is described, e.g., in Kocienski, Protecting Groups,(Thieme, 2007); Robertson, Protecting Group Chemistry, (OxfordUniversity Press, 2000); Smith et al., March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, 6^(th) Ed. (Wiley,2007); Peturssion et al., “Protecting Groups in Carbohydrate Chemistry,”J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groupsin Organic Synthesis, 4th Ed., (Wiley, 2006).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry or by chromatographic methods such as high performanceliquid chromatography (HPLC) or thin layer chromatography (TLC).

The Schemes below provide general guidance in connection with preparingthe compounds of the invention. One skilled in the art would understandthat the preparations shown in the Schemes can be modified or optimizedusing general knowledge of organic chemistry to prepare variouscompounds of the invention.

Compounds of Formula (I) can be prepared, e.g., using a process asillustrated in the schemes below.

As shown in Scheme 1, Intermediate II, which is flanked by orthogonalleaving groups, can be reacted with a nucleophilic R⁸H compound to giveintermediate III. Intermediate III is then reacted with a thiolcontaining peptide (HS—R⁷) that participates in a disulfide exchangereaction to give the final compound. Suitable leaving groups aredescribed below.

The synthesis of aminal linked compounds is shown in Scheme 2. Anucleophilic R₈H compound is reacted with a bromoacetic acid derivativeto provide intermediate IV. This ester containing intermediate ishydrolyzed under basic conditions to give intermediate acid V. Theresulting acid is transformed to the acyl azide intermediate and thensubjected to Curtius rearrangement conditions. The correspondingtransient isocyanate is trapped with hydroxy containing intermediate Ito give intermediate VII. Intermediate VII is then reacted with a thiolcontaining peptide (HS—R⁷) that participates in a disulfide exchangereaction to give the final compound.

An alternative synthesis of aminal linked conjugates is shown in Scheme3. Nucleophilic R⁸—H, which can include a protecting group (PG), isreacted with AcO-hemi-aminal carbamate VIII with a pre-installed linkedLeaving Group 2 to give Intermediate IX. This compound is treated withconditions to remove the protecting group and the resulting compound VIIis reacted with R⁷—SH to give the desired conjugate.

A further alternative synthesis of aminal linked conjugates is shown inScheme 4. Primary carbamate X with a pre-installed linked Leaving Group2 is reacted with a carbonyl compound and p-toluene sulfinate sodiumsalt to give sulfonyl carbamate XI. This is further treated withnucleophilic R⁸—H with a protecting group (PG) to give IX. This compoundis treated as previously described to give the desired conjugate.

An exemplary synthesis of thiopropionate linked conjugates is shown inScheme 5. Propionate disulfide XII with previously installed LeavingGroups 1 and 2 is reacted selectively with nucleophilic R⁸—H to giveXII. This compound is further reacted with R⁷—SH to provide the desiredconjugate.

An alternative synthesis of thiopropionate linked conjugates is shown inScheme 6. Thionoester XIV is reacted with nucleophilic R⁸—H to givepropionate thiol XV. This compound engages in a disulfide exchangereaction to provide XIII. This compound is treated with R⁷—SH to providethe desired conjugate.

The synthesis of para benzoic-linked conjugates is depicted in Scheme 7.Para amino benzoic alcohol XVI is selectively protected at the oxygen togive XVII. This is then reacted at the aniline position with II toprovide aryl carbamate XVIII. The protecting group is removed givingfree OH XIX which is treated with an activating agent to provide XXwhich contains orthogonal leaving groups. Reaction of XX with R⁸—H togive XXI followed by R⁷—SH give provides the desired conjugate.

An alternative synthesis of para benzoic-linked conjugates is shown inScheme 8. 4-Mercapto benzoic alcohol XXII is reacted in a disulfideexchange reaction to give 4-mercapto benzoic alcohol disulfide XXIcontaining Leaving Group 2. The remaining benzyl alcohol is treated toprovide activated compound XXIV. This is further reacted selectivelywith nucleophilic R⁸—H and resulting XXV with R⁷—SH to give the desiredconjugate.

The synthesis of ortho benzoic-linked conjugates is shown in Scheme 9.2-Mercapto benzoic alcohol XXVIII is reacted as previously described forScheme 8 to give the desired conjugate.

The synthesis of amino acid benzoic carbamate-linked conjugates is shownin Scheme 10. Para amino benzoic/heterobenzoic alcohol XVI can beselectively coupled to N-protected amino acid or peptide XXX to giveXXXI. The protecting group can be removed to provide XXXII which is thenreacted with II to give carbamate XXXIII with Leaving Group 2 installed.The alcohol can be selectively reacted to give carbonate XXXIV which hasLeaving Group 1 present. Leaving Group 1 can be subsequently displacedwith R⁸—H to give XXXV which is reacted with R⁷—SH to provide thedesired amino acid benzoic carbamate-linked conjugate.

The synthesis of amino acid-linked conjugates is shown in Scheme 11.N-Protected amino acid or peptide XXX can be coupled to R⁸—H to giveXXXVI. The protecting group can be removed to give XXXVII which can besubsequently reacted with II, displacing Leaving Group 1 to giveXXXVIII. This intermediate can then be treated with R⁷—SH to provide theconjugate.

The synthesis of benzoic alcohol-linked, glucuronide conjugates is shownin Scheme 12. 2-Nitro phenol XXXIX can be coupled with protectedglucuronide XL to give XLI. This intermediate can be reduced selectivelyat the carbonyl to give alcohol XLII and then the nitro group can bereduced to provide aniline XLIII. This intermediate can be furthercoupled with propionic acid derivative XLIV to give amide XLV whichcontains Leaving Group 2. XLV can then be reacted at the alcohol toinstall Leaving Group 2, giving XLVI. Leaving Group 1 can be selectivelydisplaced with R⁸—H to give XLVII. The protecting groups on theglucuronide can be removed and XLVIII can be reacted with R⁷—SH to givethe conjugate.

Cleavage of the final compound to release R⁸H can be achieved bytreating the compound with an excess of glutathione (GSH) in a bufferwith incubation at 37° C. Reversed phase HPLC analysis at a desired timecourse is used to follow the course of the cleavage.

The peptides R⁷ may be prepared using the solid-phase synthetic methodfirst described by Merrifield in J.A.C.S., Vol. 85, pgs. 2149-2154(1963), although other art-known methods may also be employed. TheMerrifield technique is well understood and is a common method forpreparation of peptides. Useful techniques for solid-phase peptidesynthesis are described in several books such as the text “Principles ofPeptide Synthesis” by Bodanszky, Springer Verlag 1984. This method ofsynthesis involves the stepwise addition of protected amino acids to agrowing peptide chain which was bound by covalent bonds to a solid resinparticle. By this procedure, reagents and by-products are removed byfiltration, thus eliminating the necessity of purifying intermediates.The general concept of this method depends on attachment of the firstamino acid of the chain to a solid polymer by a covalent bond, followedby the addition of the succeeding protected amino acids, one at a time,in a stepwise manner until the desired sequence is assembled. Finally,the protected peptide is removed from the solid resin support and theprotecting groups are cleaved off.

The amino acids may be attached to any suitable polymer. The polymermust be insoluble in the solvents used, must have a stable physical formpermitting ready filtration, and must contain a functional group towhich the first protected amino acid can be firmly linked by a covalentbond. Various polymers are suitable for this purpose, such as cellulose,polyvinyl alcohol, polymethylmethacrylate, and polystyrene.

Methods of Use

Another aspect of the present invention is the use of the compounds offormula (I) in the treatment of diseases involving acidic or hypoxicdiseased tissue, such as cancer, stroke, myocardial infarction, orlong-term neurodegenerative disease. In some embodiments, the cancer isPARP-sensitive. In some embodiments, the cancer is associated withabnormal expression or activity of ATM. In some embodiments, the canceris associated with abnormal expression or activity of ATM. In someembodiments, the cancer is associated with abnormal expression oractivity of DNA-PK. In some embodiments, the cancer is a BRCA-mutatedbreast cancer. In some embodiments, the cancer is germline BRCA-mutatedovarian cancer.

3. In these methods of treatment, a therapeutically-effective amount ofa compound of formula (I) or a pharmaceutically-acceptable salt thereofmay be administered as a single agent or in combination with other formsof therapy, such as ionizing radiation or cytotoxic agents in the caseof cancer. In combination therapy, the compound of formula (I) may beadministered before, at the same time as, or after the other therapeuticmodality, as will be appreciated by those of skill in the art. Eithermethod of treatment (single agent or combination with other forms oftherapy) may be administered as a course of treatment involving multipledoses or treatments over a period of time. Also provided herein is amethod of reducing bone marrow toxicity associated with administrationof an ionizing radiation or cytotoxic agent, which comprisesadministering to a human or other mammal a therapeutically-effectiveamount of a compound of the disclosure, or a pharmaceutically acceptablesalt thereof, in combination with the ionizing radiation or cytotoxicagent. The reduction in bone marrow toxicity can be relative to bonemarrow toxicity observed when the ionizing radiation or cytotoxic agentis administered together with R⁸H instead of the compound of theinvention having the same R⁸ (e.g., where the R⁸H compound correspondsto the protonated R⁸ moiety of the R⁸-Q-R⁷ compound of the invention).In some embodiments, toxicity can be measured by PARylation in bonemarrow tissue. In some embodiments, toxicity can be measured accordingto total nucleated bone marrow cells. In some embodiments, the cytotoxicagent can be any of those listed herein. In some embodiments, thecytoxic agent is temozolomide (TMZ).

Examples of cancers that are treatable using the compounds of thepresent disclosure include, but are not limited to, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, endometrial cancer, carcinoma of the cervix, carcinoma ofthe vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or urethra, carcinoma of the renal pelvis, neoplasm of thecentral nervous system (CNS), primary CNS lymphoma, tumor angiogenesis,spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi'ssarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers.

In some embodiments, cancers treatable with compounds of the presentdisclosure include melanoma (e.g., metastatic malignant melanoma), renalcancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormonerefractory prostate adenocarcinoma), breast cancer, triple-negativebreast cancer, colon cancer and lung cancer (e.g. non-small cell lungcancer and small cell lung cancer). Additionally, the disclosureincludes refractory or recurrent malignancies whose growth may beinhibited using the compounds of the disclosure.

In some embodiments, cancers that are treatable using the compounds ofthe present disclosure include, but are not limited to, solid tumors(e.g., prostate cancer, colon cancer, esophageal cancer, endometrialcancer, ovarian cancer, uterine cancer, renal cancer, hepatic cancer,pancreatic cancer, gastric cancer, breast cancer, lung cancer, cancersof the head and neck, thyroid cancer, glioblastoma, sarcoma, bladdercancer, etc.), hematological cancers (e.g., lymphoma, leukemia such asacute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia(CIVIL), DLBCL, mantle cell lymphoma, Non-Hodgkin lymphoma (includingrelapsed or refractory NHL and recurrent follicular), Hodgkin lymphomaor multiple myeloma) and combinations of said cancers.

In certain embodiments, a compound of formula (I) or apharmaceutically-acceptable salt thereof may be used in combination witha chemotherapeutic agent, a targeted cancer therapy, an immunotherapy orradiation therapy. The agents can be combined with the present compoundsin a single dosage form, or the agents can be administeredsimultaneously or sequentially as separate dosage forms. In someembodiments, the chemotherapeutic agent, targeted cancer therapy,immunotherapy or radiation therapy is less toxic to the patient, such asby showing reduced bone marrow toxicity, when administered together witha compound of formula (I), or a pharmaceutically acceptable saltthereof, as compared with when administered in combination with thecorresponding free DNA repair inhibiting compound (e.g., R⁸—H) such asan inhibitor of PARP, ATR, DNK-PK, or ATM as described herein.

Suitable chemotherapeutic or other anti-cancer agents include, forexample, alkylating agents (including, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes) such as uracil mustard, chlormethine, cyclophosphamide(Cytoxan™), ifosfamide, melphalan, chlorambucil, pipobroman,triethylene-melamine, triethylenethiophosphoramine, busulfan,carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

Other suitable agents for use in combination with the compounds of thepresent invention include: dacarbazine (DTIC), optionally, along withother chemotherapy drugs such as carmustine (BCNU) and cisplatin; the“Dartmouth regimen,” which consists of DTIC, BCNU, cisplatin andtamoxifen; a combination of cisplatin, vinblastine, and DTIC; ortemozolomide. Compounds according to the invention may also be combinedwith immunotherapy drugs, including cytokines such as interferon alpha,interleukin 2, and tumor necrosis factor (TNF).

Suitable chemotherapeutic or other anti-cancer agents include, forexample, antimetabolites (including, without limitation, folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors) such as methotrexate, 5-fluorouracil, floxuridine,cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,pentostatine, and gemcitabine.

Suitable chemotherapeutic or other anti-cancer agents further include,for example, certain natural products and their derivatives (forexample, vinca alkaloids, antitumor antibiotics, enzymes, lymphokinesand epipodophyllotoxins) such as vinblastine, vincristine, vindesine,bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin,idarubicin, ara-C, paclitaxel (TAXOL™), mithramycin, deoxycoformycin,mitomycin-C, L-asparaginase, interferons (especially IFN-a), etoposide,and teniposide.

Other cytotoxic agents that can be administered in combination with thecompounds of the invention include, for example, navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Also suitable are cytotoxic agents such as, for example,epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes such ascis-platin and carboplatin; biological response modifiers; growthinhibitors; antihormonal therapeutic agents; leucovorin; tegafur; andhaematopoietic growth factors.

Other anti-cancer agent(s) include antibody therapeutics such astrastuzumab (Herceptin), antibodies to costimulatory molecules such asCTLA-4, 4-1BB and PD-1, or antibodies to cytokines (IL-10, TGF-α, etc.).

Other anti-cancer agents also include those that block immune cellmigration such as antagonists to chemokine receptors, including CCR2 andCCR4.

Other anti-cancer agents also include those that augment the immunesystem such as adjuvants or adoptive T cell transfer.

Anti-cancer vaccines that can be administered in combination with thecompounds of the invention include, for example, dendritic cells,synthetic peptides, DNA vaccines and recombinant viruses.

Other suitable agents for use in combination with the compounds of thepresent invention include chemotherapy combinations such asplatinum-based doublets used in lung cancer and other solid tumors(cisplatin or carboplatin plus gemcitabine; cisplatin or carboplatinplus docetaxel; cisplatin or carboplatin plus paclitaxel; cisplatin orcarboplatin plus pemetrexed) or gemcitabine plus paclitaxel boundparticles (Abraxane®).

Compounds of this invention may be effective in combination withanti-hormonal agents for treatment of breast cancer and other tumors.Suitable examples are anti-estrogen agents including but not limited totamoxifen and toremifene, aromatase inhibitors including but not limitedto letrozole, anastrozole, and exemestane, adrenocorticosteroids (e.g.prednisone), progestins (e.g. megastrol acetate), and estrogen receptorantagonists (e.g. fulvestrant). Suitable anti-hormone agents used fortreatment of prostate and other cancers may also be combined withcompounds of the present invention. These include anti-androgensincluding but not limited to flutamide, bicalutamide, and nilutamide,luteinizing hormone-releasing hormone (LHRH) analogs includingleuprolide, goserelin, triptorelin, and histrelin, LHRH antagonists(e.g. degarelix), androgen receptor blockers (e.g. enzalutamide) andagents that inhibit androgen production (e.g. abiraterone).

Compounds of the present invention may be combined with or administeredin sequence with other agents against membrane receptor kinasesespecially for patients who have developed primary or acquiredresistance to the targeted therapy. These therapeutic agents includeinhibitors or antibodies against EGFR, Her2, VEGFR, c-Met, Ret, IGFR1,or Flt-3 and against cancer-associated fusion protein kinases such asBcr-Abl and EML4-Alk. Inhibitors against EGFR include gefitinib anderlotinib, and inhibitors against EGFR/Her2 include but are not limitedto dacomitinib, afatinib, lapitinib and neratinib. Antibodies againstthe EGFR include but are not limited to cetuximab, panitumumab andnecitumumab. Inhibitors of c-Met may be used in combination with thecompounds of the invention. These include onartumzumab, tivantnib, andINC-280. Agents against Abl (or Bcr-Abl) include imatinib, dasatinib,nilotinib, and ponatinib and those against Alk (or EML4-ALK) includecrizotinib.

Angiogenesis inhibitors may be efficacious in some tumors in combinationwith compounds of the invention. These include antibodies against VEGFor VEGFR or kinase inhibitors of VEGFR. Antibodies or other therapeuticproteins against VEGF include bevacizumab and aflibercept. Inhibitors ofVEGFR kinases and other anti-angiogenesis inhibitors include but are notlimited to sunitinib, sorafenib, axitinib, cediranib, pazopanib,regorafenib, brivanib, and vandetanib

Activation of intracellular signaling pathways is frequent in cancer,and agents targeting components of these pathways have been combinedwith receptor targeting agents to enhance efficacy and reduceresistance. Examples of agents that may be combined with compounds ofthe present invention include inhibitors of the PI3K-AKT-mTOR pathway,inhibitors of the Raf-MAPK pathway, inhibitors of JAK-STAT pathway, andinhibitors of protein chaperones and cell cycle progression.

Agents against the PI3 kinase include but are not limited topilaralisib,idelalisib, buparlisib. Inhibitors of mTOR such as rapamycin, sirolimus,temsirolimus, and everolimus may be combined with compounds of theinvention. Other suitable examples include but are not limited tovemurafenib and dabrafenib (Raf inhibitors) and trametinib, selumetiniband GDC-0973 (MEK inhibitors). Inhibitors of one or more JAKs (e.g.,ruxolitinib, baricitinib, tofacitinib), Hsp90 (e.g., tanespimycin),cyclin dependent kinases (e.g., palbociclib), HDACs (e.g.,panobinostat), PARP (e.g., olaparib), and proteasomes (e.g., bortezomib,carfilzomib) can also be combined with compounds of the presentinvention.

Methods for the safe and effective administration of most of thesechemotherapeutic agents are known to those skilled in the art. Inaddition, their administration is described in the standard literature.For example, the administration of many of the chemotherapeutic agentsis described in the “Physicians' Desk Reference” (PDR, e.g., 1996edition, Medical Economics Company, Montvale, N.J.), the disclosure ofwhich is incorporated herein by reference as if set forth in itsentirety.

The phrase “therapeutically effective amount” of a compound (therapeuticagent, active ingredient, drug, etc.) refers to an amount of thecompound to be administered to a subject in need of therapy or treatmentwhich alleviates a symptom, ameliorates a condition, or slows the onsetof disease conditions, according to clinically acceptable standards forthe disorder or condition to be treated. For instance, a therapeuticallyeffective amount can be an amount which has been demonstrated to have adesired therapeutic effect in an in vitro assay, an in vivo animalassay, or a clinical trial. The therapeutically effective amount canvary based on the particular dosage form, method of administration,treatment protocol, specific disease or condition to be treated, thebenefit/risk ratio, etc., among numerous other factors.

Said therapeutically effective amount can be obtained from a clinicaltrial, an animal model, or an in vitro cell culture assay. It is knownin the art that the effective amount suitable for human use can becalculated from the effective amount determined from an animal model oran in vitro cell culture assay. For instance, as reported by Reagan-Shawet al., FASEB J. 2008: 22(3) 659-61, “μg/ml” (effective amount based onin vitro cell culture assays)=“mg/kg body weight/day” (effective amountfor a mouse). Furthermore, the effective amount for a human can becalculated from the effective amount for a mouse based on the fact thatthe metabolism rate of mice is 6 times faster than that of humans.

As an example of treatment using a compound of formula (I) asmonotherapy, a therapeutically-effective dosage of a compound of formula(I) wherein R⁸H is a PARP inhibitor may be administered to a patientsuffering from a PARP dependent cancer, such as a female suffering fromBRCA-mutated breast cancer, germ line BRCA-mutated ovarian cancer, orfallopian tube cancer or to a patient suffering from primary peritonealcancer, squamous cell lung cancer, or non-small cell lung cancer, or toa patient suffering from stroke, myocardial infarction, or long-termneurodegenerative disease. As another example, atherapeutically-effective dosage of a compound of formula (I) whereinR⁸H is a DNA repair inhibitor targeting the protein kinaseataxia-telangiectasia mutated (ATM), the ATM-Rad3-related protein kinase(ATR), or the nuclear serine/threonine protein kinase DNA-PK could beadministered to a patient suffering from a cancer in which inhibition ofone or more of the above proteins would be therapeutically useful.

As an example of treatment using a compound of formula (I) incombination with a cytotoxic agent, a therapeutically-effective amountof a compound of formula (I) may be administered to a patient sufferingfrom cancer as part of a treatment regimen also involving atherapeutically-effective amount of ionizing radiation or a cytotoxicagent. In the context of this treatment regimen, the term“therapeutically-effective” amount should be understood to meaneffective in the combination therapy. It will be understood by those ofskill in the cancer-treatment field how to adjust the dosages to achievethe optimum therapeutic outcome.

Similarly, the appropriate dosages of the compounds of the invention fortreatment of non-cancerous diseases or conditions (such ascardiovascular diseases) may readily be determined by those of skill inthe medical arts.

The term “treating” as used herein includes the administration of acompound or composition which reduces the frequency of, delays the onsetof, or reduces the progression of symptoms of a disease involving acidicor hypoxic diseased tissue, such as cancer, stroke, myocardialinfarction, or long-term neurodegenerative disease, in a subjectrelative to a subject not receiving the compound or composition. Thiscan include reversing, reducing, or arresting the symptoms, clinicalsigns, or underlying pathology of a condition in a manner to improve orstabilize a subject's condition (e.g., regression of tumor growth, forcancer or decreasing or ameliorating myocardial ischemia reperfusioninjury in myocardial infarction, stroke, or the like cardiovasculardisease). The terms “inhibiting” or “reducing” are used for cancer inreference to methods to inhibit or to reduce tumor growth (e.g.,decrease the size of a tumor) in a population as compared to anuntreated control population.

All publications (including patents) mentioned herein are incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications, which might be used in connection with the disclosureherein described. The publications discussed throughout the text areprovided solely for their disclosure prior to the filing date of thepresent application.

Disclosed herein are several types of ranges. When a range of any typeis disclosed or claimed, the intent is to disclose or claim individuallyeach possible number that such a range could reasonably encompass,including end points of the range as well as any sub-ranges andcombinations of sub-ranges encompassed therein. When a range oftherapeutically effective amounts of an active ingredient is disclosedor claimed, for instance, the intent is to disclose or claimindividually every possible number that such a range could encompass,consistent with the disclosure herein. For example, by a disclosure thatthe therapeutically effective amount of a compound can be in a rangefrom about 1 mg/kg to about 50 mg/kg (of body weight of the subject),the intent is to recite that the therapeutically effective amount can beequal to about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg,about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg,about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg,about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg, about 31 mg/kg,about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg, about 36mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40 mg/kg,about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg, about 45mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, about 49 mg/kg,or about 50 mg/kg. Additionally, the therapeutically effective amountcan be within any sub-range included within about 1 mg/kg to about 50mg/kg (for example, the amount can be in a range from about 2 mg/kg toabout 10 mg/kg), and this also includes any combination of rangesbetween about 1 mg/kg and about 50 mg/kg (for example, the amount can bein a range from about 1 mg/kg to about 5 mg/kg or from about 20 mg/kg toabout 35 mg/kg). Likewise, all other ranges disclosed herein should beinterpreted in a manner similar to this example.

Formulation, Dosage Forms and Administration

To prepare the pharmaceutical compositions of the present invention, acompound of Formula (I) or a pharmaceutically-acceptable salt thereof iscombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques, which carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral or parenteral. In preparing the compositions in oral dosage form,any of the usual pharmaceutical media may be employed, such as forexample, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations such as for example, suspensions, elixirs, and solutions;or carriers such as starches, sugars, diluents, granulating agents,lubricants, binders, disintegrating agents, and the like in a case oforal solid preparations, such as for example, powders, capsules, andtablets. Because of their ease in administration, tablets and capsulesrepresent the most advantageous oral dosage unit form, in which casesolid pharmaceutical carriers are obviously employed. If desired,tablets may be sugar coated or enteric coated by standard techniques.For parenterals, the carrier will usually comprise sterile water,although other ingredients, for example, to aid solubility or forpreservative purposes, may be included. Injectable suspensions may alsobe prepared, in which case appropriate liquid carriers, suspendingagents, and the like may be employed. One of skill in the pharmaceuticaland medical arts will be able to readily determine a suitable dosage ofthe pharmaceutical compositions of the invention for the particulardisease or condition to be treated.

EXAMPLES

As used herein, all abbreviations, symbols and conventions areconsistent with those used in the contemporary scientific literature.See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authorsand Editors, 2nd Ed., Washington, D.C.: American Chemical Society, 1997.The following definitions describe terms and abbreviations used herein:

-   -   Brine: a saturated NaCl solution in water    -   DCM: dichloromethane    -   TFA: trifluoroacetic acid    -   DIPEA: diisopropylethylamine    -   DMA: dimethylacetamide    -   DME: dimethoxyethane    -   DMF: dimethylformamide    -   DMSO: methylsulfoxide    -   DTT: dithiothreitol    -   MSD: mass spec detector    -   Et₂O ethyl ether    -   EtOAc: ethyl acetate    -   EtOH: ethyl alcohol    -   HATU: O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HOBt: 1-hydroxybenzotriazole    -   RP: reverse phase    -   HPLC: high performance liquid chromatography    -   IPA: isopropanol    -   LAH: lithium aluminum hydride    -   N-BuLi: n-butyl lithium    -   LC-MS: liquid chromatography-mass spectrometry    -   LDA: lithium diisoproylethylamide    -   Me: methyl    -   MeOH: methanol    -   MTBE: methyl t-butyl ether    -   NMP: N-methylpyrrolidine    -   Ph: phenyl    -   PNPC: para-nitrophenylchloroformate    -   RT or rt: room temperature    -   SFC: supercritical fluid chromatography    -   TBAI: tetrabutylammonium iodide    -   TBME: tort-butylmethyl ether    -   tBu: tertiary butyl    -   THF: tetrahydrofuran    -   TEA: triethylamine    -   TMEDA: tetramethylethylenediamine    -   GSH: Glutathione    -   GS: Glutathione bonded at sulfur    -   LiOH: lithium hydroxide    -   DPPA: diphenyl phosphoryl azide    -   Sn(Bu)₂(Laurate)₂: dibutyltin dilaurate    -   PBS: phosphate buffered saline    -   ACN: acetonitrile    -   AcOH: acetic add    -   EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline    -   DMAP: 4-dimethylaminopyridine    -   EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide        HPLC Methods

All HPLC methods use reverse phase conditions: H₂O/Acetonitrile with TFAmodifier (0.05%); Flow rate: 1 mL/min; Wavelength=217 nm. Columnconditions are described below:

A: Sunfire C18 150×4.6 mm

B: Ace Equivalence 250×4.6 mm

C: Sunfire C18 150×30 mm

R⁸H Intermediates

The R⁸H groups used throughout the Examples were purchased orsynthesized as indicated in Table 2.

TABLE 2 R⁸H Intermediates Synthesis Reference or R⁸ Code R⁸H StructurePurchased Class R⁸H-1

Synthesized- Bioorganic & Medicinal Chemistry Letters 14 (2004) 2433-2437; J. Med. Chem. 2000, 43, 4084-4097 PARP R⁸H-2

Synthesized- WO0220500A2 DNA-PK R⁸H-3

Astatech 42568 PARP R⁸H-4

MedKoo 203115 PARP R⁸H-15

Chemscene CS-0937 PARP R⁸H-16

Astatech 81136 PARP R⁸H-17

MentonChem MC7030606 PARP R⁸H-18

Selleckchem S1060 PARP R⁸H-19

MedKoo 205150 PARP R⁸H-20

Advanced Chemblocks 10278 ATR R⁸H-21

Astatech 42043 ATR R⁸H-22

Medchem Express HY-10619B DNA-PK R⁸H-23

Synthesized- WO2013163190A1 DNA-PK R⁸H-24

Astatech 40964 ATM R⁸H-25

MedKoo 407142 PARP R⁸H-26

MedKoo 406357 PARP R⁸H-27

MedKoo 406362 PARP R⁸H-28

Synthesized WO2012166983A1 PARP R⁸H-29

Synthesized J. Med. Chem. 2016, 59, 6281-6292 ATM R⁸H-30

Synthesized J. Med. Chem. 2016, 59, 6281-6292 ATM R⁸H-31

ACS Med. Chem. Lett. 2018, 9, 809-814 ATM R⁸H-32

J. Med. Chem. 2018, 61, 3823-3841. ATM R⁸H-33

Synthesized ACS Med. Chem. Lett. 2015, 6, 42-46 ATR R⁸H-34

Synthesized ACS Med. Chem. Lett. 2015, 6, 37-41 ATR R⁸H-35

Oncotarget, 6, 42, 44289-44305 ATR R⁸H-36

Oncotarget, 6, 42, 44289-44305 ATRLinkers

Linkers used herein were either purchased or synthesized as shown belowin Table 3:

TABLE 3 Commerically Available Linkers Linker Synthetic Reference orCode Linker Structure Purchased L1

Alfa Aesar A15890 L2

Enamine EN300-9515 L4

Synthesized- WO2013055987A1 L5

Synthesized- WO2013055987A1 L3

Enamine EN300-220914 L6

Synthesized-ACS Med. Chem. Lett. 2016, 7, 988-993 L7

Synthesized Synlett, 2005, 20 3063 L8

Synthesized Synlett, 2005, 20 3063 L9

Synthesized Synlett, 2005, 20 3063 L10

TCI M1206 L11

Aurum GN23710 Linker XVI-1

Combiblocks OR-0735 Linker XXII-1

Combiblocks OR-5865 Linker XXVI-1

Oakwood 001239 Linker XXXI-1

Aurum DS18383

Linker L4: (2R)-2-sulfanylpropan-1-ol

Step 1: Methyl (2R)-2-acetylsulfanylpropanoate

Thioacetic acid (2.24 g, 29.4 mmol) and cesium carbonate (7.98 g, 24.5mmol) were taken up in 40 mL of dry DMF. The mixture was stirred at RTfor 30 min before the methyl (2S)-2-chloropropanoate (3.00 g, 24.5 mmol)was added. The mixture was then stirred for an additional 3 h. Diethylether (150 mL) and water (150 mL) were added and the layers separated.The aqueous layer was washed with additional ether and the combinedorganics dried over Na₂SO₄. The organic layer was concentrated and theresidue purified by column chromatography (SiO₂, 0-10% EtOAc/Hexanes) togive 3.50 g, 88% of methyl (2R)-2-acetylsulfanylpropanoate.

Step 2: (2R)-2-sulfanylpropan-1-ol

A solution of methyl (2R)-2-acetylsulfanylpropanoate (3.50 g, 21.6 mmol)in THF (30 mL) was added dropwise to a suspension of LAH (4.10 g, 108mmol) in THF (60 mL) at 0° C. After addition was complete, the mixturewas stirred at RT for 3 h. The reaction was cooled to 0° C., thenquenched by adding 2N HCl (˜75 mL) dropwise. After complete addition,the mixture was stirred at RT for 1 h. The mixture was extracted withCH₂Cl₂ (5×100 mL) and dried with Na₂SO₄. The crude(2R)-2-sulfanylpropan-1-ol, 1.60 g, 81% was carried on without furtherpurification.

Linker XXII-2: (3-methyl-4-sulfanyl-phenyl)methanol

Step 1: 4-((3-((2-ethylhexyl)oxy)-3-oxopropyl)thio)-3-methylbenzoate

To a stirred solution of methyl 4-bromo-3-methylbenzoate (0.20 g, 0.88mmol) and 2-ethylhexyl 3-mercaptopropanoate (0.21 g, 0.96 mmol) in1,4-dioxane (2 mL), DIPEA (0.32 mL, 1.80 mmol), Xanthphos (0.005 g,0.009 mmol) were added. The reaction mixture was purged with argon for 5min. Pd(dba)₃ (0.008 g, 0.009 mmol) was added and the reaction mixturewas heated at 100° C. for 6 h. After completion of the reaction, thereaction mixture was quenched with water (10 mL), and extracted intoethyl acetate (20 mL). The organic layer was washed with sodium chloridesolution (20 mL) and dried over Na₂SO₄ to afford the crude product. Thecrude product was purified by flash chromatography (SiO₂, 0-5% ethylacetate/hexane) to afford4-((3-((2-ethylhexyl)oxy)-3-oxopropyl)thio)-3-methylbenzoate as a yellowcolor liquid (0.2 g, 62% yield). MS m/z 367.1 [M+H]⁺.

Step 2: Ethyl 4-mercapto-3-methylbenzoate

A stirred solution of methyl4-((3-((2-ethylhexyl)oxy)-3-oxopropyl)thio)-3-methylbenzoate (0.20 g,0.54 mmol) in THF (5 mL) was cooled to 0° C. Sodium ethoxide (35 wt. %,2 mL) was added and the mixture was stirred for 4 h at room temperature.After completion of the reaction, the reaction mixture was acidifiedwith 2N HCl (10 mL) and concentrated. The resulting residue wasextracted into ethyl acetate (20 mL). The organic layer was washed withwater (20 mL), saturated sodium chloride solution (20 mL) and dried overNa₂SO₄ to afford ethyl 4-mercapto-3-methylbenzoate as a colorless liquid(80 mg, 80% yield). MS m/z 197.1 [M+H]⁺.

Step 3: (3-methyl-4-sulfanyl-phenyl)methanol

To a stirred solution of ethyl 4-mercapto-3-methylbenzoate (0.60 g, 3.07mmol) in THF (20 mL) maintained at 0° C. was slowly added a 1M solutionof LAH in THF (7.80 mL, 7.80 mmol). The reaction mixture was stirred atroom temperature for 3 h. After completion of the reaction, the reactionmixture was cooled to 0° C. and quenched with water (20 mL). Temperaturewas maintained below 20° C. during quenching. After completion ofquenching, the pH was adjusted to 2-3 with 2N HCl. The resulting residuewas extracted into ethyl acetate (20 mL) and the organic layer waswashed with sodium chloride solution (20 mL). The organic phase wasdried over Na₂SO₄ to afford the crude product. The crude product waspurified by flash chromatography (SiO₂, 0-20% ethyl acetate/hexane) toafford (3-methyl-4-sulfanyl-phenyl)methanol as a colorless liquid (0.45g, 90% yield). MS m/z 153.0 [M−H]⁻.

XXII-3 and XXII-4, as shown in Table 4 below, were synthesized in ananalogous manner to Linker XXII-2.

TABLE 4 Additional Linkers Linker m/z found Code Linker Structure [M −H] XXII-3

153.1 XXII-4

167.0

Intermediates Intermediate I-1: 2-(2-pyridyldisulfanyl)ethanol

To 2-(2-pyridyldisulfanyl)pyridine (4.693 g, 21.3 mmol) in 40 ml ofdegassed (N₂) MeOH was added 2-sulfanylethanol (0.498 mL, 7.10 mmol) ina drop-wise fashion. The mixture was stirred for 2-20 h under N₂. Themixture was concentrated to dryness and directly purified (SiO₂, 0-5%EtOAc/CH₂Cl₂) to give 3 fractions; (F1 mix SM A and product; F2 product;F3 2-thiopyridine/product mix). The impure material was purified again(SiO₂, 0-50% EtOAc/Hexanes) to give 1.17 g, 88% yield of2-(2-pyridyldisulfanyl)ethanol. MS m/z found 188.4 [M+H]⁺

The following Intermediates shown in Table 5 were prepared analogouslyto Intermediate I-1.

TABLE 5 Intermediates

Intermediate R¹, R² R³, R⁴ n R⁵, R⁶ X MH+ I-2  Me-H (rac) H, H 0 — H202.1 I-7  H, H H, Ph 0 — H 264.1 I-8  H, H H, 0 — H 246.1 CO₂Me I-9  H,H H, 0 — H 232.0 CH₂OMe I-10 H, H H, H 1 H, H H 367.0

Intermediate I-3: 2-methyl-2-[(5-nitro-2-pyridyl)disulfanyl]propan-1-ol

SO₂Cl₂ (0.382 mL, 4.71 mmol) was added drop-wise to a stirred suspensionof 5-nitropyridine-2-thiol (668 mg, 4.28 mmol) in dry DCM (15 mL) at 4°C. under an N₂ atmosphere. The reaction mixture turned from a yellowsuspension to a yellow solution and was allowed to warm to roomtemperature with stirring for 2 hours, after which time the mixture wasconcentrated to provide a yellow solid. The solid was re-dissolved inDCM (15 mL) and treated drop-wise with a solution of2-methyl-2-sulfanyl-propan-1-ol (454 mg, 4.28 mmol) in dry DCM (10 mL)at 4° C. under an N₂ atmosphere. The reaction mixture was allowed towarm to room temperature and stirred for 20 hours. The reaction wasmonitored by LC/MS for the desired product mass. To the mixture wasadded 50 mL of H₂O and the diluted mixture was treated with ammoniumhydroxide solution. The reaction mixture was diluted with 50 mL ofEtOAc, partitioned, and separated. The organic phase was dried withMgSO₄, filtered, and concentrated to give the crude product. The crudemixture was purified (SiO₂, 0-50% EtOAc/hexanes) to give 721 mg, 65%yield of 2-methyl-2-[(5-nitro-2-pyridyl)disulfanyl]propan-1-ol. MS m/zfound 261.7 [M+H]⁺.

Intermediate I-4: (2R)-2-(2-pyridyldisulfanyl)propan-1-ol

To 2-(2-pyridyldisulfanyl)pyridine (5.00 g, 22.7 mmol) in 40 ml of MeOHdegassed with N₂ was added (2R)-2-sulfanylpropan-1-ol (0.75 g, 8.14mmol) in a drop-wise fashion. The mixture was stirred for 2 h under N₂.The mixture was concentrated to dryness and directly loaded onto a SiO₂flash column and eluted with 0-50% EtOAc/Hexanes to give 1.17 g, 71% of(2R)-2-(2-pyridyldisulfanyl)propan-1-ol. MS m/z found 202.1 [M+H]⁺

Intermediate I-5

Intermediate I-5 was prepared in an analogous fashion to IntermediateI-4 from L-5.

Intermediate I-6

Intermediate I-6 was prepared analogously to Intermediate I-3 fromLinker L-6.

Synthesis of Intermediates XII

TABLE 6 Intermediates XII

Int. R¹, R² R³, R⁴ R⁵, R⁶ X MH+ XII-1 H, H H, H H, H H 230.0

Synthesis of Intermediate XII-1: 4-(2-pyridyldisulfanyl)butanoic acid

To 2-(2-pyridyldisulfanyl)pyridine (1120 mg, 5.08 mmol) in 20 ml ofdegassed (N₂) MeOH was added 4-sulfanylbutanoic acid (500 mg, 4.16 mmol)in a drop-wise fashion. The mixture was stirred for 16 h under N₂. Themixture was concentrated to dryness and purified by reverse-phasechromatography (Sunfire C18, 30×150 mm, 5-95% CH₃CN/H₂O, 0.05% TFA) togive 187 mg, 19.6% of 4-(2pyridyldisulfanyl)butanoic acid. MS m/z found230.0 [M+H]⁺.

Intermediate XIX-1: 2-(2-pyridyldisulfanyl)ethyl N-[4-(hydroxymethyl)phenyl]carbamate

Step 1: Synthesis of 4-[[tert-butyl(dimethyl)silyl]oxymethyl]aniline

A solution of (4-aminophenyl)methanol (5.00 g, 40.6 mmol) in anhydrousDMF (10 mL) was added dropwise within 30 min to a stirred solution oftert-butyl-chloro-dimethyl-silane (7.34 g, 48.7 mmol) and imidazole(7.90 mL, 81.2 mmol) in anhydrous DMF (40 mL). After stirring at roomtemperature for 16 h, the reaction mixture was poured into water (400mL) and extracted with CH₂Cl₂ (3×100 mL). The combined organic layerswere washed with water (2×100 mL) and dried over MgSO4. After removingthe volatiles in vacuo, the residue was purified by columnchromatography (SiO2, 0-25% EtOAc/hexanes) affording4-[[tert-butyl(dimethyl)silyl]oxymethyl]aniline (7.91 mg, 33.3 mmol,yield: 82.1%).

Step 2: Synthesis of2-(2-pyridyldisulfanyl)ethylN-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]phenyl]carbamate

To (4-nitrophenyl) 2-(2-pyridyldisulfanyl)ethyl carbonate (200 mg, 0.57mmol) in 2 mL of DMF was added4-[[tert-butyl(dimethyl)silyl]oxymethyl]aniline (202 mg, 0.85 mmol),1-hydroxybenzotriazole hydrate (104 mg, 0.68 mmol) and 200 mg ofactivated 4 A Mol sieves. The mixture was stirred for 18 h. The solidswere filtered off and the celite plug rinsed with 2 mL of DMF. Thefiltrate was concentrated to dryness and the residue purified by columnchromatography (SiO2, 0-50% EtoAc/hexanes) to afford2-(2-pyridyldisulfanyl)ethylN-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]phenyl]carbamate (244 mg,0.54 mmol, yield: 95.4%).

Step 3: 2-(2-pyridyldisulfanyl)ethyl N-[4-(hydroxymethyl)phenyl]carbamate

2-(2-Pyridyldisulfanyl)ethylN-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]phenyl]-carbamate (194 mg,0.430 mmol) was dissolved in 2 mL of THF and cooled to 0° C. under N₂.HF-pyridine (780 μL, 8.89 mmol) was added and the solution continued tostir at 0° C. for 1 h. Water (3 mL) was added, and the reaction wasneutralized with sat. NaHCO₃ and extracted with CH₂Cl₂ (3×50 mL). Theorganic layer was concentrated and purified by column chromatography(SIO₂, 0-75% EtOAc/hexane) to give2-(2-pyridyldisulfanyl)ethylN-[4-(hydroxymethyl) phenyl]carbamate (99.1mg, 0.29 mmol, yield: 68.4%).

Intermediate XXIII-1: [4-(2-pyridyldisulfanyl)phenyl]methanol

A stirred solution of 1,2-di(pyridin-2-yl)disulfane (2.68 g, 12.1 mmol)in a mixture of AcOH:ethanol (5 mL, 1:10) solvent was degassed under N₂.This was followed by addition of 4-mercaptophenyl)methanol (0.74 g, 5.2mmol) in a mixture of AcOH/ethanol (5 mL) solvent drop-wise over 20 minand stirred for 12 h under N₂ atmosphere at room temperature. Thereaction was concentrated under reduced pressure to afford the crudeproduct which is purified by column chromatography (SiO₂, 60-70%EtOAc/hexanes) to afford [4-(2-pyridyldisulfanyl)phenyl]methanol as acolourless liquid (800 mg, 61% yield).

Intermediate XXIII-2: [3-methyl-4-(2-pyridyldisulfanyl)phenyl]methanol

A stirred solution of 1,2-di(pyridin-2-yl)disulfane (1.10 g, 4.90 mmol)dissolved in acetic acid/ethanol (1:10, 37 mL) was purged with N₂ for 5min. To this, a solution of 4-mercapto-3-methylphenyl)methanol (0.51 g,3.30 mmol) in acetic acid/ethanol (1:10, 18 mL) was added drop-wise overa period of 20 min. The resulting reaction mixture was stirred for 16 hat room temperature. After completion of the reaction, the mixture wasconcentrated under reduced pressure to afford the crude product. Thecrude product was purified by flash chromatography (SiO₂, 0-70%EtOAc/hexane gradient) to afford [3-methyl-4-(2-pyridyldisulfanyl)phenyl]methanol as a colorless liquid (0.69 g, 80% yield). MSm/z found 264.0 [M+H]⁻.

Intermediates XXIII-3 and XXIII-4

Intermediates XXIII-3 and XXIII-4 as shown in Table 7 were preparedanalogously to XXIII-2.

TABLE 7 Intermediates XXIII

Intermediate R⁵, R⁶ R⁹, R¹⁰, R¹¹, R¹² MH+ XXIII-3 H, H H, H, H, Me 264.2XXIII-4 H, H Me, Me, H, H 278.0

Intermediates XXXIII from Intermediates XXXII

TABLE 8 Intermediates XXXIII Int. R₁, R₂, R₃, R₄, R₅, R₆ R₉, R₁₀, R₁₁,R₁₂ A₁, A₂ MH+ XXXIII-1 H, H, H, H H, H H, H, H, H Cit, Val 593.2

Synthesis of Intermediate XXXIII-1: 2-(2-pyridyldisulfanyl)ethylN-[(1S)-1-[[(1S)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate

To(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide(100 mg, 0.264 mmol) in 2 mL of dry DMF under N₂ is addedN,N-Diisopropylethylamine (0.122 mL, 0.659 mmol) and (4-nitrophenyl)2-(2-pyridyldisulfanyl)ethyl carbonate (92.9 mg, 0.264 mmol). Themixture is stirred for 16 h under N₂. The mixture is concentrated to asolid. The crude residue is purified on an SiO₂ column (12 g, 0-10%MeOH/CH₂Cl₂) to give 2-(2-pyridyldisulfanyl)ethylN-[(1S)-1-[[(1S)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate(149 mg, 0.251 mmol, yield: 95.4%). MS m/z found 593.2 [M+H]⁺.

Synthesis of XLI-1: Methyl(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-carboxylate

The title compound was prepared as outlined in US 2017/0145044 A1.

Synthesis of XLII-1: Methyl(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-carboxylate

The title compound was prepared as outlined in WO 2011/066418 A1.

Synthesis of XLIII-1: Methyl(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[2-amino-4-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylate

Methyl (2S, 3S, 4S,5R)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-carboxylate(361 mg, 0.74 mmol) was dissolved in MeOH and to it was added Pd/C (50.0mg, 0.47 mmol) followed by NaBH₄ (84.4 mg, 2.23 mmol). The reactionmixture was stirred at RT for 15 min. LC-MS indicated the desiredproduct had formed. The reaction mixture was filtered through celite andthen quenched with sat. NH₄Cl. The product was extracted with DCM, EtOAcand the organic layers combined. They were washed with brine andconcentrated. The crude product was partitioned in half and one portionwas purified by RP HPLC (20-95% ACN/H₂O) to give methyl(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[2-amino-4-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylate(180 mg, 0.40 mmol, 53.1% yield). MS m/z found 456.2 [M+H]⁺.

Synthesis of XLV-1: Methyl(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[3-R10-2-R11-4-(hydroxymethyl)-6-[3-(2-pyridyldisulfanyl)propanoylamino]phenoxy]tetrahydropyran-2-carboxylate

EEDQ (130 mg, 0.527 mmol) was added to 3-(2-pyridyldisulfanyl)propanoicacid (56.7 mg, 0.263 mmol) dissolved in DCM and was stirred for 20 minunder N₂. The methyl(2S,3S,4S,5R)-3,4,5-triacetoxy-6-[2-amino-4-(hydroxymethyl)phenoxy]tetrahydropyran-2-carboxylate(120 mg, 0.263 mmol) was dissolved in DCM and added to the mixture andstirred overnight. LC-MS indicated the desired product had been formed.The reaction mixture was concentrated and purified by reverse phasechromatography (20-95% ACN/H₂O) to give 63 mg of methyl(2S,3S,4S,5R)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-[3-(2-pyridyldisulfanyl)propanoylamino]phenoxy]tetrahydropyran-2-carboxylate(63.0 mg, 0.0965 mmol, yield: 36.6%). MS m/z found 653.2 [M+H]⁺.

Intermediate II-1: (4-nitrophenyl) 2-(2-pyridyldisulfanyl)ethylcarbonate

To 2-(2-pyridyldisulfanyl)ethanol (517 mg, 2.76 mmol) in 20 ml CH₂Cl₂under N₂ at 4° C. was added N,N-Diisopropylethylamine (1.02 mL, 5.52mmol) and (4-nitrophenyl) carbonochloridate (834 mg, 4.14 mmol). Themixture was stirred for 16 h under N₂. The mixture was dissolved into 40mL of EtOAc and quenched with 20 mL of sat. NH₄Cl. The mixture waswashed with 2×20 mL H2O and 1×20 mL sat. brine. The crude mixture waspurified via SiO₂ column, eluting from 0-50% EtOAc/hexanes to give(4-nitrophenyl) 2-(2-pyridyldisulfanyl)ethyl carbonate (482 mg, 50%yield).

Intermediates II-2, II-3, and II-5

Intermediates II-2, II-3, and II-5 were prepared analogously toIntermediate II-1 using the appropriate intermediates I-2, I-3, and I-5,as shown below:

TABLE 9 Additional Intermediates

Intermediate R¹, R² R³, R⁴ n R⁵, R⁶ X MH+ II-2 Me-H (rac) H, H 0 — H367.1 II-5 H, Me H, H 0 — H 367.1 II-3 Me, Me H, H 0 — NO₂

Intermediate II-4: (4-nitrophenyl)[(2R)-2-(2-pyridyldisulfanyl)propyl]carbonate

To (2R)-2-(2-pyridyldisulfanyl)propan-1-ol (0.39 g, 1.94 mmol) in THFunder N₂ was added pyridine (0.16 mL, 1.94 mmol) and the (4-nitrophenyl)carbonochloridate (0.59 g, 2.91 mmol). The mixture was stirred for 16 hunder N₂. The mixture was diluted with EtOAc and quenched with 20 mL ofsat. NH₄Cl. The mixture was washed with water and brine and the organiclayer concentrated. The crude mixture was purified by columnchromatography (SiO₂, 0-50% EtOAc/Hexanes) to afford 0.59 g, 83% of(4-nitrophenyl) [(2R)-2-(2-pyridyldisulfanyl)propyl]carbonate. MS m/zfound 367.1 [M+H]⁺.

Intermediates II-6 Through II-9

Intermediates II-6, II-7, II-8, and II-9 were prepared analogously toIntermediate II-4, as shown below in Table 10:

TABLE 10 Additional Intermediates

Intermediate R¹, R² R³, R⁴ n R⁵, R⁶ X MH+ II-6  —CH₂CH₂CH₂— H, H 0 — NO₂438.0 (R¹ and R² form a cyclobutyl) II-7  H, H H, Ph 0 — H 429.1 II-8 H, H H, CO₂Me 0 — H 411.0 II-9  H, H H, 0 — H 397.1 CH₂OMe II-10 H, H H,H 1 H, H H 495.1

Synthesis of Intermediate XXVII-1

Intermediate XXVII-1 is prepared analogously to Intermediates XXII.

TABLE 11 Intermediates XXVII

Int. R^(l), R², R³, R⁴ R⁵, R⁶ MH+ XXVII-1 H, H, H, H H, H 250.1

Intermediate XX-1: (4-nitrophenyl)[4-[2-(2-pyridyldisulfanyl)ethoxycarbonyl amino]phenyl]methyl carbonate

To 2-(2-pyridyldisulfanyl)ethyl N-[4-(hydroxymethyl)phenyl]carbamate(136 mg, 0.41 mmol) in 5 ml CH₂Cl₂ under N₂ was added DIPEA (0.15 mL,0.81 mmol) and (4-nitrophenyl) carbonochloridate (122 mg, 0.61 mmol).The mixture was stirred for 16 h under N₂. The mixture was dissolvedinto 20 mL of EtOAc and quenched with 20 mL of sat. NH₄C₁. The mixturewas washed with 2×20 mL H₂O and 1×20 mL sat. brine. The crude mixturewas purified via column chromatography (SiO₂, 0-25% EtOAc/hexanes) toafford (4-nitrophenyl)[4-[2-(2-pyridyldisulfanyl)ethoxycarbonylamino]phenyl]methyl carbonate(44.2 mg, 0.09 mmol, yield: 21.8%).

Intermediate XXIV-1: (4-nitrophenyl)[4-(2-pyridyldisulfanyl)phenyl]methyl carbonate

To a stirred solution of (4-(pyridin-2-yldisulfanyl)phenyl)methanol(0.40 g, 1.60 mmol) in CH₂Cl₂ (10 mL) were added 4-nitrophenylchloroformate (0.65 g, 3.2 mmol), pyridine (0.25 mL, 3.20 mmol),catalytic amount of DMAP (0.005 g) at 0° C. The mixture was allowed tostir for 2 h at room temperature. The reaction mixture was quenched with1.5 N HCl solution. The organic layer was separated and washed withbrine, dried over anhydrous Na₂SO₄ and concentrated. The crude productwas purified by column chromatography (SiO₂, 20-30% of EtOAc/hexanes) toafford (4-nitrophenyl) [4-(2-pyridyldisulfanyl)phenyl]methyl carbonateas a colourless liquid (600 mg, 91% yield); MS m/z 415.0 [M+H]⁺

Intermediate XXIV-2: [3-methyl-4-(2-pyridyldisulfanyl)phenyl]methyl(4-nitrophenyl) carbonate

To a stirred solution of(3-methyl-4-(pyridin-2-yldisulfaneyl)phenyl)methanol (0.69 g, 2.60 mmol)dissolved in CH₂Cl₂ (10 mL) was added 4-nitrophenyl chloroformate (1.05g, 5.2 mmol), pyridine (0.43 mL, 5.2 mmol), and a catalytic amount ofDMAP (0.005 g) at 0° C. The mixture was allowed to stir for 2 h at roomtemperature. After completion of the reaction, the reaction mixture wasquenched with 1.5 N HCl. The organic layer was separated, washed withsodium chloride solution (10 mL), dried over anhydrous Na₂SO₄ andconcentrated. The crude product was purified by flash columnchromatography (SiO₂, 20-30% EtOAc/hexanes) to afford[3-methyl-4-(2-pyridyldisulfanyl)phenyl]methyl (4-nitrophenyl) carbonate(700 mg, 62% yield). MS m/z 429.0 [M+H]⁺.

The following intermediate were prepared analogously to IntermediatesXXIV-1 and XXIV-2 as shown below in Table 12:

TABLE 12 Additional Intermediates

Intermediate R⁵, R⁶ R⁹, R¹⁰, R¹¹, R¹² X MH+ XXIV-3 H, H H, H, Me, H H429.0 XXIV-4 H, H Me, Me, H, H H 443.0

Intermediates XXVIII

TABLE 13 Intermediates XXVIII

Int. R¹, R², R³, R⁴ R₅, R₆ MH+ XXVIII-1 H, H, H, H H, H 415.0

Intermediate XXVIII-1 was prepared analogously to Intermediates XXIV.

Intermediates XXXIV

TABLE 14 Intermediates XXXIV

Int. R¹, R², R³, R⁴ R⁵, R⁶ R⁹, R¹⁰, R¹¹, R¹² A₁, A₂ MH+ XXXIV-1 H, H, H,H H, H H, H, H, H Cit, Val 758.2

Synthesis of XXXIV-1:[4-[[(2S)-2-[[(2S)-3-methyl-2-[2-(2-pyridyldisulfanyl)-ethoxycarbonylamino]butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl(4-nitrophenyl) carbonate

2-(2-pyridyldisulfanyl)ethylN-[(1S)-1-[[(1S)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate(149 mg, 0.251 mmol) was dissolved into 2 mL of dry DMF and cooled to 4°C. To this is added bis(4-nitrophenyl) carbonate (153 mg, 0.503 mmol)and N,N-Diisopropylethylamine (0.0928 mL, 0.503 mmol). The mixture wasallowed to warm to RT over 2 h. The progress of the reaction wasmonitored by LC-MS. The mixture was concentrated and purified (25 gSiO₂, 0-10% MeOH/CH2Cl2) to give[4-[[(2S)-2-[[(2S)-3-methyl-2-[2-(2-pyridyldisulfanyl)ethoxycarbonylamino]butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl(4-nitrophenyl) carbonate (139 mg, yield: 73.1%). MS m/z found 758.2[M+H]⁺.

Synthesis of XLVI-1: Methyl(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[2-R11-4-[(4-nitrophenoxy)carbonyloxymethyl]-6-[3-(2-pyridyldisulfanyl)propanoylamino]-phenoxy]tetrahydropyran-2-carboxylate

Methyl(2S,3S,4S,5R)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-[3-(2-pyridyldisulfanyl)propanoylamino]phenoxy]tetrahydropyran-2-carboxylate(102 mg, 0.15 mmol) was dissolved in THF and to it was added pyridine(0.02 mL, 0.19 mmol) followed by (4-nitrophenyl) carbonochloridate (63.0mg, 0.313 mmol). The reaction mixture was stirred under N₂ overnight.LC-MS indicated a complete reaction. The mixture was diluted with 100 mLEtOAc and quenched with 50 mL of sat. NH₄Cl. The mixture was washed with50 mL sat. brine. The organic layer was dried with NaSO₄ andconcentrated. The crude mixture was purified by column chromatography(50-100% EtOAc/Hexanes) to give (58.0 mg, 0.07 mmol, 45.4%). MS m/zfound 818.2 [M+H]⁺.

Intermediate III-1: [4-(4-carbamoyl-1H-benzimidazol-2-yl)phenyl]methyl2-(2-pyridyldisulfanyl)ethyl carbonate

To 2-[4-(hydroxymethyl)phenyl]-1˜{H}-benzimidazole-4-carboxamide (150mg, 0.561 mmol) in 4 mL of dry DMF under N₂ was addedN,N-diisopropylethylamine (0.207 mL, 1.12 mmol), DMAP (68.6 mg, 0.561mmol), and (4-nitrophenyl) 2-(2-pyridyldisulfanyl)ethyl carbonate (241mg, 0.684 mmol). The mixture was stirred for 16 h., and then dilutedwith 50 ml of EtOAc and successively washed with 1×20 mL of sat. NH₄Cl,3×30 mL of sat. NaHCO₃, 3×30 mL of H₂O, and 1×20 mL of sat. brine. Theorganic phase was dried with MgSO₄, filtered, and concentrated. Thecrude residue was purified (SiO₂, 50-100% EtOAc/hexanes) to give 260 mg,97% yield of[4-(4-carbamoyl-1H-benzimidazol-2-yl)phenyl]methyl2-(2-pyridyldisulfanyl)ethylcarbonate.

Intermediate III-4: [(2R)-2-(2-pyridyldisulfanyl)propyl]N-[[4-(6-fluoro-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamate

To a mixture of HOBt (48.0 mg, 0.31 mmol), pyridine (0.11 mL, 1.31mmol), finely ground molecular sieve 4 Å (250 mg), and the6-fluoro-2-[4-(methylaminomethyl)phenyl]-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-9-onephosphate (110 mg, 0.26 mmol) in 5 mL of anhydrous DMF was added the(4-nitrophenyl) [(2R)-2-(2-pyridyldisulfanyl)propyl]carbonate[Intermediate II-4] (105 mg, 0.29 mmol). After stirring for 16 h at roomtemperature, the molecular sieves were filtered off and the solventremoved in vacuo. The residue was then adsorbed onto SiO₂ and purifiedby column chromatography (SiO₂, 0-10% MeOH/CH₂Cl₂) to afford[(2R)-2-(2-pyridyldisulfanyl)propyl]N-[[4-(6-fluoro-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamate(115 mg, 80% yield) MS m/z 551.1 (M+H)⁺.

Intermediates III-2, III-3, and III-5 Through III-15

The following intermediates were prepared analogously to IntermediatesIII-1 and III-4 as shown below in Table 15.

TABLE 15 Additional Intermediates

Inter- mediate R⁸H R¹, R² R³, R⁴ n R⁵, R⁶ X MH+ III-2  R⁸H-1 Me-H (rac)H, H — H 495.1 III-3  R⁸H-1 Me, Me H, H — NO₂ III-5  R⁸H-1 Me, H H, H —H 496.1 III-6  R⁸H-2 H, H H, H — H 451.1 III-7  R⁸H-3 H, H H, H — H534.2 III-8  R⁸H-4 H, H H, H — H 458.1 III-9  R⁸H-16 H, H H, H — H 536.4III-10 R⁸H-16 H, Me H, H — H 551.1 III-11 R⁸H-16 Me, H H, H — H 551.1III-12 R⁸H-16 —CH₂CH₂CH₂— H, H — NO₂ 622.2 III-13 R⁸H-16 H, H Ph, H — H613.2 III-14 R⁸H-16 H, H CO₂Me• H — H 595.2 III-15 R⁸H-16 H, H CH₂OMe —H 581.2 III-16 R₈H-3 H, Me H, H 0 — H 548.1 III-17 R₈H-3 Me, H H, H 0 —H 548.1 III-18 R₈H-4 H, Me H, H 0 — H 472.1 III-19 R₈H-4 Me, H H, H 0 —H 472.1 III-20 R₈H16 H, H H, H 1 H, H H 495.2Ester Linked Intermediates XIII

TABLE 16 Intermediates XIII

Int. R⁸ R¹, R² R³, R⁴ R⁵, R⁶ X MH+ XIII-1 R⁸H-1 H, H H, H H, H H 479.0

Synthesis of XIII-1: [4-(4-carbamoyl-1H-benzimidazol-2-yl)phenyl]methyl4-(2-pyridyldisulfanyl)butanoate

To a mixture of 4-(2-pyridyldisulfanyl)butanoic acid TFA salt (81.2 mg,0.236 mmol), EDC HCl (47.8 mg, 0.250 mmol), and DIEA (0.0986 mL, 0.576mmol) in 2 mL of DMF was added2-[4-(hydroxymethyl)phenyl]-1H-benzimidazole-4-carboxamide (51.3 mg,0.192 mmol). The mixture was stirred overnight and monitored by LC-MS.The mixture was concentrated and purified with SiO₂ chromatography(0-10% MeOH/CH2Cl2) to give (83.1 mg, 0.17 mmol, yield: 90.4%) MS m/zfound 479.0 [M+H]⁺.

Intermediate VII-1: 2-(2-pyridyldisulfanyl)ethylN-[[(11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2-methyl-1,2,4-triazol-3-yl)-4-oxo-2,3,10-triazatricyclo[7.3.1.05,13]trideca-1,5,7,9(13)-tetraen-10-yl]methyl]carbamate

The2-[(11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2-methyl-1,2,4-triazol-3-yl)-4-oxo-2,3,10-triazatricyclo[7.3.1.05,13]trideca-1,5,7,9(13)-tetraen-10-yl]acetylazide, V-1, (170 mg, 0.367 mmol) was dissolved in dry DMF (2 mL) and2-(2-pyridyldisulfanyl)ethanol (137 mg, 0.734 mmol) is added. Thereaction was heated to 65° C. for 2 h. Catalytic dibutyltin dilaurate(40 uL) was added and the reaction mixture stirred overnight at 65° C.The mixture was concentrated and purified by column chromatography(0-10% MeOH/DCM) to give 3 peaks. NMR indicated peak 2 was the desiredproduct. MALDI showed 3 masses of 624 (desired product), 646(product+23) and 380 (BMN). Yield: 80 mg of Intermediate VII-1:2-(2-pyridyldisulfanyl)ethylN-[[(11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2-methyl-1,2,4-triazol-3-yl)-4-oxo-2,3,10-triazatricyclo[7.3.1.05,13]trideca-1,5,7,9(13)-tetraen-10-yl]methyl]carbamate.

Intermediates VII-2 Through VII-5

Intermediates VII-2, VII-3 and VII-4 were prepared using R₈H-15 andintermediates II-4, II-5 and II-6, respectively. Intermediate VII-5 wasprepared using R₈H-17 and intermediate II-1 as shown in Table 17.

TABLE 17 Additional Intermediates

Int. R⁸H R¹, R² R³, R⁴ R⁵, R⁶ X MH+ VII-2 R⁸H-15 H, Me H, H H, H H 637.2VII-3 R⁸H-15 Me, H H, H H, H H 637.2 VII-4 R⁸H-15 —CH₂CH₂CH₂— H, H H, HNO₂ 708.2 (R1 and R2 form a cyclopropyl) VII-5 R⁸H-17 H, H H, H H, H H541.1

Intermediate VII-6: 2-(2-pyridyldisulfanyl)ethylN-[[6-fluoro-2-[4-(methylaminomethyl)phenyl]-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-3-yl]methyl]carbamate

Step 1: (((2-(pyridin-2-yldisulfanyl)ethoxy)carbonyl)amino)methylacetate

To a stirred solution of 2-(pyridin-2-yldisulfanyl)ethyl carbamate (0.10g, 0.43 mmol) in acetic acid (0.59 mL) was added paraformaldehyde (0.01g, 0.47 mmol) and acetic anhydride (1.78 mL). The mixture was heated at75° C. for 3 h. After completion of reaction, the reaction was quenchedwith water (20 mL) and extracted with ethyl acetate (20 mL×2). Thecombined organic layer was washed with water (20 mL) and brine solution(20 mL). The organic phase was dried over anhydrous Na₂SO₄ andconcentrated. The crude product was purified by column chromatography(SiO₂, 40-50% of ethyl acetate/hexanes) to afford(((2-(pyridin-2-yldisulfanyl)ethoxy)carbonyl)amino)methyl acetate as ayellow liquid (120 mg, 70%). MS m/z 303.3 (M+H)⁺

Step 2:(9H-fluoren-9-yl)methyl(4-(8-fluoro-1-oxo-6-((((2-(pyridin-2yldisulfanyl)ethoxy)carbonyl)amino)methyl)-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5-yl)benzyl)(methyl)carbamate

To a stirred solution of (9H-fluoren-9-yl)methyl(4-(8-fluoro-1-oxo-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5-yl)benzyl)(methyl)carbamate(0.05 g, 0.09 mmol) in acetone (1.0 mL) was added cesium carbonate (0.06g, 0.18 mmol) and stirred for 5 min at room temperature under N₂atmosphere. Then(((2-(pyridin-2-yldisulfanyl)ethoxy)carbonyl)amino)methyl acetate (0.03g, 0.09 mmol) and DMF (0.1 mL) were added and further stirred for 30 minat room temperature. The reaction was quenched with water (25 mL) andthen extracted with 10% of MeOH/CH₂Cl₂ mixture (50 mL×2). The combinedorganic layer was dried over anhydrous sodium sulphate and concentratedunder reduced pressure to afford the crude product which was purified byflash column chromatography using 60-70% of EtOAc/hexanes mixture toafford(9H-fluoren-9-yl)methyl(4-(8-fluoro-1-oxo-6-((((2-(pyridin-2yldisulfanyl)ethoxy)carbonyl)amino)methyl)-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5-yl)benzyl)(methyl)carbamate(12 mg, 17% yield); MS m/z 788.8 (M+H)⁺.

Step 3: 2-(2-pyridyldisulfanyl)ethylN-[[6-fluoro-2-[4-(methylaminomethyl)phenyl]-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-3-yl]methyl]carbamate

(9H-fluoren-9-yl)methyl(4-(8-fluoro-1-oxo-6-((((2-(pyridin-2yldisulfanyl)ethoxy)carbonyl)amino)methyl)-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5-yl)benzyl)(methyl)carbamate(0.15 g, 0.19 mmol) was stirred with 20% of diethylamine in DMF (1.4 mL)for 1 h under N₂ at room temperature. The reaction mixture wasconcentrated to dryness and purified by prep-HPLC [Column: Inertsil ODS3V (250 mm×20 mm X 5 mic); Mobile phase-A-0.1% ammonia in H₂O: Mobilephase-B-ACN] to afford 2-(2-pyridyldisulfanyl)ethylN-[[6-fluoro-2-[4-(methylaminomethyl)phenyl]-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-3-yl]methyl]carbamateas a colorless solid (30 mg, 28% yield). MS m/z 566.4 (M+H)⁺.

Intermediate VII-7: 2-(2-pyridyldisulfanyl)ethylN-[1-[6-fluoro-2-[4-(methylaminomethyl)phenyl]-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-3-yl]-3-methyl-butyl]carbamate

Step 1: 2-(pyridin-2-yldisulfanyl)ethyl(3-methyl-1-(phenylsulfonyl)butyl)carbamate

To a stirred solution of 2-(pyridin-2-yldisulfanyl)ethyl carbamate (2.50g, 0.01 mol) in THF:water (19 mL, 1:1) were added sodiump-toluenesulfinate (1.93 g, 0.01 mol), 3-methylbutanal (1.26 mL, 0.01mol) and then formic acid (2.5 mL). The mixture was stirred for 16 h atroom temperature under N₂. The reaction mixture was concentrated todryness and then purified by flash column chromatography (SiO₂, 40-70%EtOAc/hexanes) to afford 2-(pyridin-2-yldisulfanyl)ethyl(3-methyl-1-(phenylsulfonyl)butyl)carbamate (1.7 g, 41% yield). MSm/z 455.1 [M+H]⁺.

Step 2: (9H-fluoren-9-yl)methyl(4-(8-fluoro-6-(3-methyl-1-(((2-(pyridin-2-yldisulfanyl) ethoxy)carbonyl)amino)butyl)-1-oxo-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5-yl)benzyl)(methyl)carbamate

To a stirred suspension of(9H-fluoren-9-yl)methyl(4-(8-fluoro-1-oxo-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5yl)benzyl)(methyl)carbamate(0.5 g, 0.92 mmol) and cesium carbonate (0.89 g, 2.74 mmol) inCH₂Cl₂:DMF (5:1, 12 mL) was added 2-(pyridin-2-yldisulfanyl)ethyl(3-methyl-1-(phenylsulfonyl)butyl)carbamate (0.80 g, 1.83 mmol)portion wise over 4 h. The mixture was stirred an additional 2 h underN₂ at room temperature. The reaction was monitored by TLC. The reactionmixture was diluted with water (25 mL) and DCM (100 mL). The organiclayer was separated, washed with water, brine. The organic phase wasdried over anhydrous Na₂SO₄ and concentrated. The crude product waspurified by flash column chromatography (SiO₂, 90-100% EtOAc/hexanes) toafford (9H-fluoren-9-yl)methyl(4-(8-fluoro-6-(3-methyl-1-(((2-(pyridin-2-yldisulfanyl) ethoxy)carbonyl)amino)butyl)-1-oxo-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5-yl)benzyl)(methyl)carbamate (0.35 g, 45% yield). MS m/z 844.1 [M+H]⁺.

Step 3: 2-(2-pyridyldisulfanyl)ethylN-[1-[6-fluoro-2-[4-(methylaminomethyl)phenyl]-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-3-yl]-3-methyl-butyl]carbamate

(9H-fluoren-9-yl)methyl(4-(8-fluoro-6-(3-methyl-1-(((2-(pyridin-2-yldisulfanyl) ethoxy)carbonyl)amino)butyl)-1-oxo-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-5-yl)benzyl)(methyl)carbamate (0.30 g, 0.355 mmol) was stirred with 10% piperidine in DMF(1.4 mL) for 20 min at room temperature under N₂. The reaction mixturewas concentrated to dryness and then triturated with diethyl ether toobtain a colorless solid. The resultant solid was further purified byprep HPLC [Column: Inertsil ODS 3V (250 mm×20 mm X 5 mic), Mobilephase-A-0.1% ammonia in H₂O: Mobile phase-B-ACN] to afford2-(2-pyridyldisulfanyl)ethylN-[1-[6-fluoro-2-[4-(methylaminomethyl)phenyl]-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-3-yl]-3-methyl-butyl]carbamate(0.07 g, 30% yield). MS m/z 622.0 [M+H]⁺.

Intermediate XXI-1: [4-[2-(2-pyridyldisulfanyl)ethoxycarbonylamino]phenyl]methylN-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamate

To2-[4-(methylaminomethyl)phenyl]-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7-tetraen-9-one;phosphoric acid (36.0 mg, 0.09 mmol) in 2 mL of dry DMF under N₂ wasadded DIPEA (0.03 mL, 0.18 mmol), DMAP (10.9 mg, 0.09 mmol) and(4-nitrophenyl) [4-[2-(2-pyridyldisulfanyl)ethoxycarbonylamino]phenyl]methyl carbonate (44.8 mg, 0.09 mmol). The mixture wasstirred for 16 h. The mixture was diluted with 20 ml of EtOAc, washedwith 1×20 mL of sat. NH₄Cl, 2×20 mL of sat. NaHCO₃, 3×30 mL of H₂O and1×20 mL of sat. brine. The mixture was dried with MgSO₄, filtered andconcentrated. The crude residue was purified by column chromatography(SiO₂, 0-5% MeOH/CH₂Cl₂) to give [4-[2-(2-pyridyldisulfanyl)ethoxycarbonylamino]phenyl]methylN-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamate(44.2 mg, 0.06 mmol, yield: 75.4%).

Intermediate XXV-1: [4-(2-pyridyldisulfanyl)phenyl]methylN-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamate

To a stirred solution of2-[4-(methylaminomethyl)phenyl]-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7-tetraen-9-one;phosphoric acid (1.00 g, 3.09 mmol) in THF (20 mL) under N₂ was addedTEA (1.40 mL, 3.04 mmol), HOBt (0.21 g, 1.50 mmol) and 4-nitrophenyl(4-(pyridin-2-yldisulfaneyl)benzyl) carbonate (1.40 g, 3.40 mmol). Themixture was stirred under N₂ for 16 h at room temperature. The reactionmixture was concentrated and the crude purified by flash chromatography(SiO₂, 0-5% MeOH/CH₂Cl₂ to afford [4-(2-pyridyldisulfanyl)phenyl]methylN-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamateas a colourless solid (1.13 g, 59% yield). MS m/z 599.0 (M+H)⁺.

Intermediates XXV-2 Through XXV-5

Intermediates XXV-2 through XXV-5 were prepared analogously toIntermediate XXV-1 using the appropriate R₈—H compound and intermediatesXXIV-2 through XXIV-5 as shown below in Table 18.

TABLE 18 Additional Intermediates

R⁹, R¹⁰, Int. R⁸H R⁵, R⁶ R¹¹, R¹² X MH+ XXV-2 R⁸H-16 H, H Me, H, H, H H613.2 XXV-3 R⁸H-16 H, H H, H, Me, H H 613.0 XXV-4 R⁸H-16 H, H Me, Me, H,H H 627.0 XXV-5 R₈H-3 H, H H, H, H, H H 596.2 XXV-6 R₈H-4 H, H H, H, H,H H 520.2 XXV-7 R₈H-21 H, H H, H, H, H H 739.2

Synthesis of Intermediates XXVIX from XXVII

TABLE 19 Intermediates XXVIX

Int. R⁸ R¹, R² R³, R⁴ R⁵, R⁶ X MH+ XXVIX-1 R₈H-16 H, H H, H H, H H 599.1

Intermediate XXVIX-1 was prepared from Intermediate XXVIII-1 analogouslyto Intermediate XXV-1.

Intermediates XXXV from XXXIV

TABLE 20 Intermediates XXXV

Int. R⁸ R¹, R², R³, R⁴, R⁵, R⁶ R⁹, R₁₀, R₁₁, R₁₂ A₁, A₂ MH+ XXXV-1R₈H-16 H, H, H, H H, H H, H, H, H Cit, Val 942.4

Intermediate XXXV-1 was prepared from Intermediate XXXIV-1 analogouslyto Intermediate XXV-1.

Intermediates XXXVII from XXX

TABLE 21 Intermediates XXXVII

Int. R⁸ R¹, R², R³, R⁴, A₁, A₂ X MH+ XXXVII-1 R⁸H-16 H, H, H, H Pro, GlyH 478.2

Synthesis of XXXVII-1:(2S)-1-(2-aminoacetyl)-N-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-pyrrolidine-2-carboxamide

Step 1. Synthesis oftert-butyl-N-[2-[(2S)-2-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl-methyl-carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]carbamate

1-[2-(tert-Butoxycarbonylamino)acetyl]pyrrolidine-2-carboxylic acid(0.16 g, 0.59 mmol) was dissolved in DMF and to it was added1-hydroxybenzotriazole hydrate (80.0%, 114 mg, 0.59 mmol) and EDC HCl(114 mg, 0.59 mmol). The solution was stirred at RT for 15 min beforethe6-fluoro-2-[4-(methylaminomethyl)phenyl]-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-9-one;phosphoric acid (200 mg, 0.475 mmol) and N,N-diisopropylethylamine (0.44mL, 2.37 mmol) were added. The solution was then heated to 65° C.overnight. LC-MS indicated a complete reaction. The reaction mixture wasdiluted with EtOAc, washed with sat. NH₄Cl, water, and brine. The crude[1-[2-(tert-butoxycarbonylamino)acetyl]pyrrolidin-2-yl]N-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamate(141 mg, 0.24 mmol, yield: 50.0%) was carried on as is. MS m/z 478.2(M+H minus BOC)⁺.

Step 2. Synthesis of(2S)-1-(2-aminoacetyl)-N-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-pyrrolidine-2-carboxamide

Thetert-Butyl-N-[2-[2-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl-methyl-carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]carbamate(141 mg, 0.24 mmol) was dissolved in DCM and 1 mL HCl (4.00 M, 0.12 mL,0.48 mmol) in dioxane was added. The reaction mixture was stirred at RTovernight. LC-MS indicated a complete reaction. The reaction mixture wasconcentrated and purified by reverse phase chromatography (20-85%ACN/H₂O). to give 68 mg of1-(2-aminoacetyl)-N-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-pyrrolidine-2-carboxamide(68.0 mg, 0.142 mmol, yield: 58.3%). MS m/z 478.2 (M+H)⁺.

Intermediates XXXVIII

TABLE 22 Intermediates XXXVIII

Int. R⁸ R₁, R₂, R₃, R₄, A₁, A₂ X MH+ XXXVIII-1 R₈H-16 H, H, H, H Pro,Gly H 691.2

Synthesis of XXXVIII-1: 2-(2-pyridyldisulfanyl)ethylN-[2-[(2S)-2-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl-methyl-carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]carbamate

To1-(2-aminoacetyl)-N-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-pyrrolidine-2-carboxamide(68.0 mg, 0.142 mmol) in 2 mL of dry DMF was added DMAP (17.4 mg, 0.142mmol), (4-nitrophenyl) 2-(2-pyridyldisulfanyl)ethyl carbonate (50.2 mg,0.142 mmol) and N,N-diisopropylethylamine (27.6 mg, 0.214 mmol). Themixture stirred for 16 h and then diluted with 50 ml of EtOAc, washedwith 1×20 mL of sat. NH₄Cl, 3×30 mL of H₂O and 1×20 mL of sat. brine.The mixture was dried with MgSO₄, filtered and concentrated. The cruderesidue was purified on an SiO₂ column (0-3% MeOH/DCM) to give 30 mg of2-(2-pyridyldisulfanyl)ethylN-[2-[2-[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl-methyl-carbamoyl]pyrrolidin-1-yl]-2-oxo-ethyl]carbamate(30.0 mg, 0.0434 mmol, yield: 30.5%). MS m/z 691.2 (M+H)⁺.

Intermediates XLVIII from XLVI

TABLE 23 Intermediates XLVIII

R₁, R₂, R₅, R₉, R₁₀, Int. R8 R₃, R₄, R₆ R₁₁, R₁₂ X MH+ XLVII-1 R₈H- H,H, H, H, H, H 1002.1 16 H, H H H, H

Synthesis of XLVII-1: Methyl(2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[3-R10-2-R11-4-[[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl-methyl-carbamoyl]oxymethyl]-6-[3-(2-pyridyldisulfanyl)propanoylamino]phenoxy]tetrahydropyran-2-carboxylate

To a mixture of 1-hydroxybenzotriazole hydrate (13.0 mg, 0.0851 mmol),finely ground molecular sieve 4 Å (100 mg), and6-fluoro-2-[4-(methylaminomethyl)phenyl]-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-9-one;phosphoric acid (35.9 mg, 0.0851 mmol) in 2 mL of anhydrous DMF wasadded methyl(2S,3S,4S,5R)-3,4,5-triacetoxy-6-[4-[(4-nitrophenoxy)carbonyloxymethyl]-2-[3-(2-pyridyldisulfanyl)propanoylamino]phenoxy]tetrahydropyran-2-carboxylate(58.0 mg, 0.0709 mmol). After stirring for 16 h at room temperature, themolecular sieve was filtered off and the solvent was removed in vacuo.The reaction mixture was diluted with EtOAc, washed with sat. NH₄Cl,water and brine. The organic layer was dried with NaSO₄ andconcentrated. The crude residue was purified by column chromatography(0-3% MeOH/DCM) to give 35 mg of methyl(2S,3S,4S,5R)-3,4,5-triacetoxy-6-[4-[[[4-(6-fluoro-9-oxo-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl-methyl-carbamoyl]oxymethyl]-2-[3-(2-pyridyldisulfanyl)propanoylamino]phenoxy]tetrahydropyran-2-carboxylate(43.0 mg, 0.0429 mmol, yield: 60.5%) with a slight unknown impurity. MSm/z 1002.1 (M+H)⁺.

Conjugated Compounds

Example 3

One 8 mL vial containing PBS and one 8 mL vial containing DMF weredegassed with bubbling N₂ for 1 h. In a separate vial was placed PeptideVariant 3 (“Pv3,” 50.0 mg, 1.31e-5 mol). The degassed 1.0 mL of PBS anda 3.0 mL portion of DMF were added to[4-(4-carbamoyl-1H-benzimidazol-2-yl)phenyl]methyl2-(2-pyridyldisulfanyl)ethyl carbonate (18.9 mg, 3.92e-5 mol). To themixture was added CH₃CO₂H (0.05 mL 0.000873 mol). The mixture was placedunder N₂ and stirred for at RT for 16 h. The mixture was monitored forprogress with RP HPLC until complete consumption of the starting peptideand remaining pyridyl disulfide (Ace Equivalence 250×4.6 mm, 50%isocratic, 25 min run). The crude reaction mixture was purified bypreparative RP HPLC (Sunfire 30×150 mm; CH₃CN/H₂O (0.1% TFA) gradient,16 min run) to give 28.6 mg, 54% yield of Example 3.

Example 18

Two separate 8 mL vials containing PBS and DMF, respectively, weredegassed with bubbling N₂ for 1 h. In a separate vial was placed Pv1(50.0 mg, 0.02 mmol). To this vial was added[(2R)-2-(2-pyridyldisulfanyl)propyl]N-[[4-(6-fluoro-3,10-diazatricyclo[6.4.1.04,13]trideca-1,4,6,8(13)-tetraen-2-yl)phenyl]methyl]-N-methyl-carbamate[Intermediate III-4] (16.2 mg, 0.03 mmol), 1.33 mL of PBS and 4 mL ofDMF followed by acetic acid (4.2 μL, 0.08 mmol). The mixture was placedunder N₂ and stirred at RT overnight. The mixture was purified by prepHPLC (40-72% CH₃CN/H₂O, 15 min) to give 29.0 mg, 0.008 mmol, 53% ofexample 18.

The following compounds were prepared analogously to Example 18 usingthe appropriate Intermediates and peptides.

TABLE 24 Example Compounds MS Column A: Maldi- % ACN/H₂O TOF Run TimeExample Structure B: m/z = 3 RT  1

A: 3649.5 A 25-75%   10 min  6.9 min  2

A: 4381.8 A 25-75%   10 min  7.7 min  3

A: 4081.8 B 20-80%   15 min 15.6 min  4

A: 4094.2 Diastereomer 1 A 45-55%   10 min  6.3 min Diastereomer 2 A45-55%   10 min  6.6 min  5

A: 4107.7 A 30-50%   10 min  6.6 min  6

A: 4095.0 A 45-55%   10 min  6.3 min  7

A: 4094.7 A 45-55%   10 min  6.6 min  8

A: 4350.8 A 50% iso   10 min  5.5 min  9

A: 4049.6 A 25-75%   10 min  8.5 min 10

A: 4133.0 A 50% iso   10 min  6.5 min 11

A: 4055.3 A 25-75%   10 min  6.7 min 15

A: 4436.2 A 10-95%   10 min  9.1 min 18

A: 3718.3 C 40-80%   15 min  7.4 min 19

A: 4450.3 C 45-71%   11 min  7.9 min 20

A: 4147.2 C 40-80%   15 min  6.9 min 21

A: 3718.6 C 30-45%   15 min  6.0 min 22

A: 4449.8 C 45-71%   11 min  8.3 min 23

A: 4148.9 C 40-72%   12 min  7.0 24

A: 4477.3 C 30-50%   13 min  6.3 min 25

A: 4511.8 C 30-50%   11 min  7.5 min 26

A: 4493.8 C 48-64%   11 min  8.0 min 27

A: 4478.6 C 52-60%   11 min  8.5 min 44

A: 3744.3 C 50-85%   13 min  4.4 min 45

A: 4145.3 C 25-95%   15 min  8.5 min 46

A: 4146.0 C 25-95%   15 min  8.5 min 47

A: 4446.5 C 35-95%   12 min  7.3 min 48

A: 4446.5 C 35-95%   12 min  7.3 min 49

A: 4434.2 C 35-91%   11 min  7.2 min 51

A: 4069.7 C 20-65%   10 min  8.2 min 52

A: 4070.3 C 20-65%   10 min  8.2 min 53

A: 4357.6 C 35-91%   12 min  7.3 min 54

A: 4370.6 C 35-70%   11 min  7.4 min 55

A: 4369.9 C 35-70%   11 min  7.4 min 57

B: 1617.1 C 25-75%   15 min 11.5 min 58

B: 1542.1 C 25-75%   15 min 11.7 min 61

B: 1465.5 C 40-64%   10 min  6.3 min

The following compounds were prepared analogously to Example 18 from theappropriate VII Intermediates and peptides.

TABLE 25 Example Compounds MS Column A: Maldi- % ACN/H₂O TOF Run TimeExample Structure B: m/z = 3 RT 12

A: 4522.3 A 40-80%  8.2 min 13

A: 4536.5 A 40-80%   10 min  8.4 min 14

A: 4535.7 A 40-80%   10 min  8.4 min 16

A: 3790.0 A 40-80%   10 min  7.1 min 17

A: 4220.1 A 40-80%  10 min  6.7 min 33

A: 4462.8 C 40-58%   9 min  6.8 min 34

A: 4520.7 Diastereomer 1 C 40-67%   12 min  9.8 min 35

A: 4521.4 Diastereomer 2 C 40-57%   12 min 10.2 min 38

A: 4440.5 C 40-60%   12 min  8.5 min 39

A: 4559.6 C 50-80%   15 min  6.5 min 40

A: 3804.0 C 40-62%   12 min  8.6 min 41

A: 4234.7 C 40-70%   11 min  6.5 min 42

A: 3805.3 C 40-62%   12 min  8.5 min

Synthesis of the Final Compounds from Intermediates XII

The following compound was synthesized from Intermediate XII-1.

TABLE 26 Additional Examples MS Column A: Maldi- % ACN/H₂O TOF Run TimeExample Structure B: m/z = 3 RT 60

B: 1460.8 C 20-65%   2 min 11.1 min

Example 32

Example 32 was synthesized from Intermediate XXI and peptide Pv2. The MS(Maldi-TOF) found was 4582.9. The compound was purified using 30-50%acetonitrile in water, Conditions 3, eluting at 11 and 8.3 min.

Synthesis of Compounds from Intermediates XXV

The following compounds were synthesized analogously to Example 18 fromIntermediates XXV.

TABLE 27 Example Compounds MS A: Maldi- Column TOF % ACN/H₂O ExampleStructure B: m/z = 3 RT 28

A: 4497.8 C 50-80%  15 min 6.5 min 29

A: 4510.9 C 50-100%  20 min 7.4 min 30

A: 4510.3 C 50-100%  20 min 7.3 min 31

A: 4526.2 C 50-100%  20 min 7.0 min 36

A: 3767.3 C 40-90%  15 min 7.0 37

A: 4196.8 C 40-90%  15 min 6.4 min 50

B: 1397.6 C 20-95%  16 min 9.5 min 56

B: 1373.5 C 35-60%  15 min 9.3 min

Synthesis of the Final Compounds from Intermediates XXV

The following compound was synthesized analogously to Example 18 fromIntermediate XXV.

TABLE 28 Example Compounds MS A: Maldi- Column TOF % ACN/H₂O ExampleStructure B: m/z = 3 RT 59

B: 1499.8 C 50-92%  16 min 6.1 min

Synthesis of the Final Compounds from Intermediates XXXV

The following compound was synthesized analogously to Example 18 fromIntermediate XXXV-1.

TABLE 29 Example Compounds MS A: Maldi- Column TOF % ACN/H₂O ExampleStructure B: m/z = 3 RT 62

1614.4 C 40-68%  10 min 8.4 min

Synthesis of the Final Compounds from Intermediates XXXVIII

The following compound was synthesized analogously to Example 18 fromIntermediates XXXVIII-1.

TABLE 30 Example Compounds MS A: Maldi- Column TOF % ACN/H₂O ExampleStructure B: m/z = 3 RT 64

B: 1531.8 C 35-75%  10 min 7.6 min

Example A. Cleavage of Compounds

To(2S)-2-amino-5-[[(1R)-2-(carboxymethylamino)-2-oxo-1-(sulfanylmethyl)ethyl]amino]-5-oxo-pentanoic acid (4.90 mg, 0.0159 mmol) was added 2 mL of 1MTris HCl buffer (pH 7.0) to create an 8 mM solution. An aliquot of 1 mLof this solution was added to Example 3 (0.400 mg, 9.81×10⁻⁵ mmol) tocreate a 100 μM solution. This mixture was heated at 37° C. with timepoints taken at 15 minute intervals to measure for conjugate integrity.A steady loss of conjugate is observed with commensurate appearance of2-[4-(hydroxymethyl)phenyl]-1˜{H}-benzimidazole-4-carboxamide. Thecleavage was complete by the 60 min time point as observed/confirmed byMSD. HPLC conditions: ES Industries Sonoma 4.6×50 mm; 5-100% CH₃CN/H₂O(0.1% TFA); 5.5 min run Conjugate RT: 3.71 Product RT: 2.24.

Other compounds were similarly cleaved to give the appropriate R₈Hmolecules.

The crude cleavage solution containing the cleaved compounds of theinvention was assayed as described below in Examples B and C to assessthe amount of cleaved R⁸H. In addition to the cleaved compounds, assayswere also performed on several of the R⁸ starting materials set out inTable 2.

Example B. Enzymatic Assay 1 of Cleaved Compounds (PARP)

Analysis was performed using Trevigen's HT F Homogeneous PARP InhibitionAssay Kit (#4690-096-K) as follows: Purified PARP enzyme was incubatedin the presence of a serially diluted PARP inhibitor (e.g., a compoundof the Examples cleaved according to Example A) in duplicate and 1 uMNAD for thirty minutes at room temperature in black, round-bottom96-well plates. An equal volume of cycling mix was added to each welland the reaction was incubated for one hour at room temperature. Thereactions were stopped with the addition of Stop Solution and the plateswere read on a BioTek Cytation 5 plate reader using the fluorescence(544 nm excitation and 590 nm emission) endpoint. Results were plottedin GraphPad Prism and are given below.

TABLE 31 HT F Homogeneous PARP Inhibition Assay of Cleaved CompoundsIC₅₀ Enzymatic Assayed Compound Assay 1 (nM) R⁸H-1 96.8 Example 3(cleaved) 104 Example 6 (cleaved) 202 Example 7 (cleaved) 111

Example C. Enzymatic Assay 2 of Cleaved Compounds (PARP)

Analysis was performed using Trevigen's HT Universal ChemiluminescentPARP Assay Kit (#4676-096-K) as follows: Purified PARP enzyme wasincubated in the presence of a serially diluted PARP inhibitor (e.g., acompound of the Examples cleaved according to Example A) in duplicatefor ten minutes at room temperature in rehydrated 96-well stripwellscontaining bound histones. An equal volume of 1×PARP cocktail containingactivated DNA was added to each well and the reaction was incubated forone hour at room temperature. The wells were washed twice with1×PBS+0.1% Triton X-100 and twice with PBS. 50 ul/well of dilutedstreptavidin-HRP was added and the wells were incubated for one hour atroom temperature. The wells were washed twice with 1×PBS+0.1% TritonX-100 and washed twice with PBS. The liquid was removed, 100 ul/well 1:1PeroxyGlow A/PeroxyGlow B was added and the chemiluminescent readingswere measured on a BioTek Cytation 5 plate reader using the luminescencefiber endpoint. Results were plotted in GraphPad Prism and are shownbelow.

TABLE 32 HT Universal Chemiluminescent PARP Assay of Cleaved CompoundsIC₅₀ Enzymatic Assayed Compound Assay 2 (nM) R⁸H-1 6.4 R⁸H-3 3.9 R⁸H-41.3 R⁸H-15 1.0 R⁸H-16 0.8 R⁸H-17 1.0 Example 1 (Cleaved) 12.1 Example 2(Cleaved) 9.52 Example 3 (Cleaved) 9.15 Example 6 (Cleaved) 9.29 Example7 (Cleaved) 6.45 Example 10 (Cleaved) 64.9 Example 11 (Cleaved) 1.08Example 12 (Cleaved) 0.47 Example 13 (Cleaved) 1.0 Example 14 (Cleaved)0.5 Example 15 (Cleaved) 6.47 Example 16 (Cleaved) 0.61 Example 17(Cleaved) 0.41 Example 18 (Cleaved) 1.1 Example 19 (Cleaved) 2.2 Example20 (Cleaved) 1.7 Example 21 (Cleaved) 2.2 Example 22 (Cleaved) 1.8Example 23 (Cleaved) 2.5 Example 24 (Cleaved) 7.6 Example 25 (Cleaved)3.4 Example 26 (Cleaved) 2.0 Example 27 (Cleaved) 3.9 Example 28(Cleaved) 2.4 Example 29 (Cleaved) 6.4 Example 30 (Cleaved) 2.9 Example31 (Cleaved) 3.3 Example 32 (Cleaved) 43.9 Example 33 (Cleaved) 2.3Example 34 (Cleaved) 2.5 Example 35 (Cleaved) 3.1 Example 36 (Cleaved)2.2 Example 37 (Cleaved) 3.1 Example 38 (Cleaved) 2.8 Example 39(Cleaved) 3.0 Example 40 (Cleaved) 1.1 Example 41 (Cleaved) 1.9 Example42 (Cleaved) 1.4 Example 43 (Cleaved) 1.4 Example 44 (Cleaved) 1.5Example 45 (Cleaved) 3.2 Example 46 (Cleaved) 3.7 Example 47 (Cleaved)2.4 Example 48 (Cleaved) 2.1 Example 49 (Cleaved) 26 Example 50(Cleaved) 2.0 Example 51 (Cleaved) 0.9 Example 52 (Cleaved) 1.1 Example53 (Cleaved) 1.0 Example 54 (Cleaved) 0.6 Example 55 (Cleaved) 0.7Example 56 (Cleaved) 0.8

Example D. Elisa Parylation Assay

Twelve-well tissue culture plates were seeded with HeLa cells andincubated at 37° C. in 5% CO₂ to yield 80% confluent cell monolayers thefollowing day. The monolayers were treated with a three-fold dilutionseries of either free drug or conjugate at a desired pH for 1 hour at37° C. The monolayers were aspirated and each well received 150 ul ofRIPA lysis buffer supplemented with protease and phosphatase inhibitors.Plates were incubated on ice for 10 minutes and then frozen. Uponthawing, the lysates were supplemented with Mg and incubated with DNAsefor 90 minutes at 37° C. The samples were clarified by centrifugation at12,000×g for 5′ at 4° C. and the cleared lysate was transferred to aclean tube and protein determinations were performed using a BCA ProteinAssay.

Analysis was performed using Trevigen's HT PARP in vivo PharmacodynamicAssay II (#4520-096-K) as follows: Duplicate 25 ul sample lysates wereloaded on pre-coated/pre-blocked ELISA stripwells and incubated for 16hours at 4° C. in tandem with serially diluted purified PAR standards.The wells were washed 4× with PBST and incubated with 50 ul/well of PARpolyclonal detecting antibody in antibody diluent for 2 hours at roomtemperature. The wells were washed 4× with PBST and incubated with 50ul/well Goat anti-rabbit IgG-HRP conjugate in antibody diluent for 1hour at room temperature. The wells were washed 4× with PBST, incubatedwith 100 ul/well of 1:1 PARP PeroxyGlow A and PARP PeroxyGlow B and readin a BioTek Cytation 5 using the luminescence fiber endpoint. Resultswere plotted as raw luminescence units or as PAR (pg/ml) using valuescalculated from the standard curve and are presented below.

TABLE 33 Parylation Assay Results IC₅₀ Elisa IC₅₀ Elisa IC₅₀ Elisa IC₅₀Elisa PARP PARP PARP PARP Inhibi- Inhibi- Inhibi- Inhibi- tion(nM)tion(nM) tion(nM) tion(nM) Example pH 5.5 pH 6.2 pH 7.4 pH 8.0 1 115 5872 93, 135 1290, 1400 3 151 616 6 239 456 7 282 542 10 785 3075 11 IC9102 12 101 159 186 116Cells are treated as previously described at pH 7.4 and incubated for 16h.

TABLE 34 Parylation Assay Results IC50 Elisa PARP IC50 Elisa PARPInhibition(nM) pH 7.4 Inhibition(nM) pH 7.4 Compound (BRCA−/−) DLD-1cells, 16 h SW620 cells, 16 h 18 2401 19 2381 20 2196 2767 21 3545 223877 23 1425 28 712 2197 29 1314 30 497 31 1140 32 4995 34 2092 35 158036 271 1174 37 439 1386 40 522 41 459 42 540 43 380 51 4178 54 1857 552928 56 103

Example E. Growth Delay Assay

Cells were plated in 96 well black walled-clear bottom plates (Griener),DLD-1 WT cells at 2500 cells per well, DLD-1 BRCA2−/− at 5000 cells perwell, in growth media containing 10% FBS. Cells were allowed to adhereat room temperature for 60 minutes before returning to a 37 C, 5% CO2incubator. After 24 hours, media was removed and replaced with freshgrowth media containing various drug concentrations. Each drugconcentration was added in triplicate. Non-drug treated controlscontained growth media only. Cells were returned to the incubator.Ninety-six hours after addition of drug, cells were fixed with 4%paraformaldehyde for 20 minutes and stained with Hoechst at 1 ug/mL. Theplates were imaged on a Cytation 5 auto imager (BioTek) and cells werecounted using CellProfiler (http://cellprofiler.org). The percent cellgrowth delay was calculated and data plotted using GraphPad Prism.

TABLE 35 Growth Delay Assay Summary IC₅₀ Growth Delay (nM) pH 7.4, BRCA(−/−) Example DLD-1, 96 h 12 162 13 51 14 72 16 84 17 50 39 104 40 64 4143 42 53 43 46

The above results demonstrate that the compounds of the invention arecleaved to release free R⁸H when subjected to conditions replicatingthose within a cell and exhibit inhibition of the biological target,which in this particular assay is PARP. Based on these results one ofskill in the art would readily recognize the compounds of the inventionto be useful for treatment of diseases involving acidic or hypoxicdiseased tissue, and particularly for treating PARP mutated cancers.

Example F. In Vivo Tumor Growth Delay

Study Design (R⁸H-15, Example 12)

Female Nude mice arrived at the facility at 6 weeks of age and werehoused 5 per cage on Alpha-Dri bedding in a disposable IVC caging system(Innovive). After an acclimation period of 5-10 days, DLD-1 BRACA2^(−/−)cells were diluted 1:1 in Phenol Red-free Matrigel and subcutaneouslyimplanted into the left flank of each mouse at a density of 5×10⁶ cellsin 100 μL. Xenograft tumor growth was monitored and caliper measurementswere obtained twice weekly. When xenografts reached a minimal volume of100 mm³, mice were administered an intraperitoneal (IP) dose of vehicle,Example 12 (6.4, 20 or 50 mg/kg), or an oral dose of R⁸H-15 (0.3 mg/kg)once daily for 8 days. All mice were administered an oral dose of 10mg/kg temozolomide (TMZ) prepared in 20% glucose immediately afterdosing. Caliper measurements were obtained twice weekly to evaluatecompound effect on tumor growth. Monitoring of tumor growth continuedafter the 8-day dosing period for another 7 weeks (wash out period).Mice were euthanized if body weight loss exceeded 20% or if tumor volumeincreased to 4× the original size. Kaplan-Meier analysis was used toevaluate survival rate based on death or removal from study.

Study Design (R⁸H-16, Example 18)

Female Nude mice arrived at the facility at 6 weeks of age and werehoused 5 per cage on Alpha-Dri bedding in a disposable IVC caging system(Innovive). After an acclimation period of 5-10 days, DLD-1 BRACA2^(−/−)cells were diluted 1:1 in Phenol Red-free Matrigel and subcutaneouslyimplanted into the left flank of each mouse at a density of 5×10⁶ cellsin 100 μL. Xenograft tumor growth was monitored and caliper measurementswere obtained twice weekly. When xenografts reached a minimal volume of100 mm³, mice were administered an intraperitoneal (IP) dose of vehicle,Example 18 (8.8, 17.7 or 44.2 mg/kg), or R⁸H-16 (1, 2, 5 mg/kg), oncedaily for 8 days. All mice were administered an oral dose of 10 mg/kgtemozolomide (TMZ) prepared in 20% glucose immediately after dosing for5 days. Caliper measurements were obtained twice weekly to evaluatecompound effect on tumor growth. Monitoring of tumor growth continuedafter the 8-day dosing period for another 8-day (wash out period). Micewere euthanized if body weight loss exceeded 20% or if tumor volumeincreased to 4× the original size.

DLD-1 BRCA2^(−/−) Cell Preparation for Implantation (R⁸H-15, Example 12;R⁸H-16,

Example 18

Matrigel was thawed overnight on ice at 4° C. prior to prepare for DLD-1BRCA2^(−/−) cell implantation and was kept on ice during all preparatorysteps. Cells were passaged between one and three days prior topreparation for implantation. Growth medium was replaced every 2-3 daysas needed to maintain cell viability. On the day of implantation, cellswere trypsinized, washed with complete media and pelleted bycentrifugation at 1200 rpm for 5 minutes. The supernatant was decantedand cells were washed three times with sterile PBS and pelleted bycentrifugation. During the final centrifugation, viability wasdetermined using trypan blue exclusion. Cells were resuspended insterile PBS at a concentration of 5×10⁶ cells/50 μL. Prior toimplantation, cells were mixed 1:1 with Matrigel for a finalconcentration of 5×10⁶ cells/100 μL. The Matrigel/cell mixture was kepton ice in a conical tube until used for implantation.

DLD-1 BRCA2^(−/−) Cell Implantation (R⁸H-15, Example 12; R⁸H-16, Example18)

Matrigel/DLD-1 BRCA2^(−/−) cells (5×10⁶ cells/100 μL) kept on ice in aconical tube were drawn into sterile 1 cc syringes fitted with sterile27-gauge needles. Excess Matrigel/cell mixture was expelled back intothe conical tube leaving an injection volume of 100 μL in each syringe.Filled syringes were kept on ice until time of implantation to avoidpolymerization of the Matrigel in the syringe. Cells were subcutaneouslyimplanted into the left flank of each mouse and upon the development ofpalpable xenografts, caliper measurements were obtained twice weekly.Compound evaluation proceeded when xenografts reached a minimal volumeof 100 mm³.

Compound Administration (R⁸H-15, Example 12)

Intraperitoneal doses of 6.4, 20 and 50 mg/kg Example 12 were preparedin 8% PEG400+2% Tween 80 in PBS as described above and were administeredonce daily for 8 days. Mice were dosed at a volume of 12 mL/kg (300 μLper 25 g mouse). Oral doses of 0.3 mg/kg R⁸H-15 (BMN673) were preparedin 0.5% methyl cellulose and were administered once daily for 8 days atvolume of 10 mL/kg (250 μL per 25 g mouse) for comparison. Immediatelyafter compound administration, all mice were administered an oral doseof 10 mg/kg temozolomide (TMZ) in 20% glucose.

Compound Administration (R⁸H-16, Example 18)

Example 18 was dissolved in 100% dimethyl sulfoxide (DMSO) to yield a0.1 mg/μL stock solution. Intraperitoneal (IP) doses of 8.83, 17.66 or44.15 mg/kg were prepared fresh daily by diluting 14, 29 or 72 μL of thestock, respectively, with 1950 μL of a vehicle comprised of 8% PEG400+2%Tween 80 in PBS. The doses were vortexed to achieve homogenoussuspensions and were administered at a concentration of 12 mg/mL (300 μLper 25 g mouse), once daily for 8 days. Each day, 2.5 mg of R⁸H-16 wassuspended in 6 mL of a vehicle comprised of 8% PEG400+2% Tween 80 inPBS. The suspension was sonicated for 10 minutes to result in ahomogeneous 5 mg/kg intraperitoneal dose which was further diluted 1 to5 and 1 to 2.5 with vehicle to result in 1 and 2 mg/kg doses,respectively. All doses were vortexed and administered at aconcentration of 12 mL/kg (300 μL per 25 g mouse) once daily for 8 days.Oral doses of 50 mg/kg temozolomide (TMZ) were prepared fresh daily bysuspending 70 mg of compound in 14 mL of a 20% glucose vehicle. Thefinal 5 mg/mL doses were sonicated for 15 minutes until homogenoussuspensions were achieved. Mice were orally administered TMZ at 10 mL/kg(250 μL per 25 g mouse) once daily for 5 days immediately followingadministration of Example 18 or R⁸H-16.

Tumor Growth

Tumor growth was monitored and caliper measurements were obtained twiceweekly over a dosing period of 8 days to evaluate compound efficacyagainst tumor growth rate. Growth measurements continued for another 7weeks after dosing ceased. Mice were removed from study when tumorvolume exceeded 4× its original size.

Statistical Analysis

Analysis of variance (ANOVA) was used to test for significantdifferences between groups. Post-hoc Bonferroni multiple comparison testanalysis was used to determine significant differences among means. Allstatistical analysis was accomplished using Graph Pad Prism 7.03software. Kaplan-Meier analysis was used to evaluate survival rate basedon death or removal from study when body weight loss exceeded 20% frominitial body weight.

FIG. 1 shows the tumor growth delay of R⁸H-15 and Example 12 inBRCA^(−/−) Mice.

FIG. 2 shows survival of R⁸H-15 and Example 12 in BRCA^(−/−) Mice.

FIG. 3 shows the tumor growth delay of R⁸H-16 and Example 18 inBRCA^(−/−) Mice.

Example G. Tumor Parylation

Study Design (R⁸H-16, Example 18)

Female Nude mice arrived at the facility at 6 weeks of age and werehoused 5 per cage on Alpha-Dri bedding in a disposable IVC caging system(Innovive). Human DLD-1 BRACA2^(−/−) cells were diluted 1:1 in PhenolRed-free Matrigel and subcutaneously implanted into the left flank ofeach mouse at a density of 5×10⁶ cells in 100 μL. Caliper measurementswere obtained twice weekly and treatment began when xenografts reached aminimal volume of 250 mm³. Intraperitoneal (IP) doses of Example 18(8.83, 17.66 or 44.15 mg/kg) or R⁸H-16 (2 or 10 mg/kg) were administeredonce daily for 9 days in an 8% PEG400+2% Tween 80 vehicle. Mice werealso administered an oral dose of 20% glucose. The eighth dose wasadministered in the evening about 12 hours before the ninth dose. Serumand tissue samples were collected 2 hours after administration of theninth dose. Compound effect against PARylation was determined in tumorand bone marrow.

Compound Administration (R⁸H-16, Example 18)

Example 18 was dissolved in 100% dimethyl sulfoxide (DMSO) to yield a0.1 mg/μL stock solution. Intraperitoneal (IP) doses of 8.8, 17.7 or44.2 mg/kg were prepared fresh daily by diluting 13, 26 or 66 μL of thestock, respectively, with 1800 μL of a vehicle comprised of 8% PEG400+2%Tween 80 in PBS. The doses were vortexed to achieve homogenoussuspensions. Intraperitoneal (IP) doses of 10 mg/kg R⁸H-16 were preparedfresh daily by suspending 5 mg of R⁸H-16 in 6 mL of a vehicle comprisedof 8% PEG400+2% Tween 80 in PBS. The doses were vortexed to achievehomogenous suspensions. IP doses of 2 mg/kg were prepared by dilutingthe 10 mg/kg dose 1:5 with vehicle. Intraperitoneal (IP) doses ofExample 18 or R⁸H-16 were administered once daily for 9 days at a volumeof 12 mL/kg (300 μL, per 25 g mouse).

Tissue Collection (R⁸H-16, Example 18)

Following blood collection, mice were euthanized by cervical dislocationunder anesthesia. Xenograft tumors were removed, weighed, and cut intosmaller pieces with a scalpel blade. Random 100 mg tumor samples werecollected in cryo-tubes, snap frozen in liquid nitrogen and stored at−80° C. until processed. PARylation measurements were made in tumorhomogenates.

PARylation in Tumor Homogenates (R⁸H-16, Example 18)

Frozen tumor samples were homogenized in a RIPA buffer containingprotease and phosphatase inhibitors (1 mg/mL) and 300 μL aliquots werebrought up to a 1% SDS final concentration. Samples were heated at 100°C. for 5 minutes, quenched on ice and clarified by centrifugation at14,000×g for 5 minutes. Tumor samples were diluted 1:10 in RIPA/HALTbuffer for protein quantification using a Pierce Rapid Gold BCA ProteinAssay kit. Samples were diluted to 200 μg/mL (10 μg/well) in samplebuffer and 50 μL aliquots (10 m/well) were loaded onpre-coated/pre-blocked ELISA strip wells. Following a 16-hour incubationat 4° C., samples were washed 4× with PBST (1 L PBS+1 mL Tween 20) andincubated with 50 μL PAR detecting antibody for 2 hours at roomtemperature. Samples were washed 4× with PBST and incubated with 50 μLgoat anti-rabbit IgG-HRP conjugate for 1 hour at room temperature.Following a final 4× wash with PBST, 100 μL of PeroxyGlow™ was added toeach sample and luminescence was determined on a BioTek Cytation 5 usingluminescence fiber endpoint and strip well area plate definition.

FIG. 4 shows the effect of 9-Day intraperitoneal administration ofExample 18 and R⁸H-16 on tumor PARylation in a murine DLD-1 BRCA2^(−/−)xenograft model.

Example H. Bone Marrow Selectivity

Study Design (R⁸H-15, Example 12)

Female Nude mice arrived at the facility at 6 weeks of age and werehoused 5 per cage on Alpha-Dri bedding in a disposable IVC caging system(Innovive). After an acclimation period of 5-10 days, DLD-1 BRACA2^(−/−)tumor cells were diluted 1:1 in Phenol Red-free Matrigel andsubcutaneously implanted into the left flank of each mouse at a densityof 5×10⁶ cells in 100 μL. Tumor growth was monitored and calipermeasurements were obtained twice weekly. When tumors reached a minimalvolume of 100 mm³, mice were administered an oral dose of 50 mg/kgTemozolomide (TMZ) alone or in combination with either an oral dose of0.3 mg/kg R⁸H-15 or an intravenous dose of 10 mg/kg Example 12, oncedaily for 2 days. On Day 3, fed mice were euthanized by cervicaldislocation under anesthesia and tumors were removed, weighed and snapfrozen in liquid nitrogen. Femurs were removed and bone marrow wasextruded with PBS into 50 mL conical tubes for isolation of bone marrowcells by centrifugation. Myelotoxicity was evaluated by bone marrow cellcount and by PARylation inhibition as determined by Elisa. Compoundeffectiveness against tumor cell viability was determined by an ElisaPARylation assay.

Study Design (R⁸H-16, Example 18)

Female Nude mice arrived at the facility at 6 weeks of age and werehoused 5 per cage on Alpha-Dri bedding in a disposable IVC caging system(Innovive). Human DLD-1 BRACA2^(−/−) cells were diluted 1:1 in PhenolRed-free Matrigel and subcutaneously implanted into the left flank ofeach mouse at a density of 5×10⁶ cells in 100 μL. Caliper measurementswere obtained twice weekly and treatment began when xenografts reached aminimal volume of 250 mm³. Intraperitoneal (IP) doses of Example 18(8.83, 17.66 or 44.15 mg/kg) or R⁸H-16 (2 or 10 mg/kg) were administeredonce daily for 9 days in an 8% PEG400+2% Tween 80 vehicle. Mice werealso administered an oral dose of 20% glucose. The eighth dose wasadministered in the evening about 12 hours before the ninth dose. Serumand tissue samples were collected 2 hours after administration of theninth dose. Compound effect against PARylation was determined in bonemarrow.

Compound Administration (R⁸H-15, Example 12)

5 mg of Example 12 was dissolved in 50 μL of 100% dimethyl sulfoxide(DMSO) to yield a 0.1 mg/μL stock solution. The final 10 mg/kgintravenous dose was prepared by diluting 25 μL of the stock with 2475μL of sterile PBS. The dose was gently mixed to result in a clearinjectable 1 mg/mL dosing solution. Doses were administered once dailyfor 2 days by tail vein injection at 10 mL/kg (200 μL per 20 g mouse).Mice were dosed in combination with an oral 50 g/kg dose ofTemozolomide. An oral dose of 3 mg/kg R⁸H-15 was prepared by suspending1.5 mg of compound in 5 mL of a 10% Dimethyl Acetamide (DMAc)+6%Solutol+84% PBS vehicle. The suspension was further diluted 1:10 in thevehicle. The final 0.3 mg/kg dose was sonicated for 15 minutes until ahomogenous 0.3 mg/mL suspension was achieved. Mice were orally dosed at10 mL/kg (200 μL per 20 g mouse) once daily for 2 days in combinationwith an oral 50 mg/kg dose of Temozolomide.

Compound Administration (R⁸H-16, Example 18)

Compound administration was as described in the Compound Administration(R⁸H-16, Example 18) section of Example F.

Bone Marrow Collection (R⁸H-15, Example 12)

Following tumor collection, femurs were removed and bone marrow wasextruded into 50 mL conical tubes by flushing the bones with a 23-gaugeneedle fitted on a 5 cc syringe containing RPMI+2% fetal bovine serum(FBS). Bone marrow was homogenized by gentle pipetting and filteredthrough 100 μm nylon mesh filters. Bone marrow cells were pelleted bycentrifugation at 1200 rpm for 5 minutes at 4° C. Cells were washed with5 mL of PBE (PBS+0.2% bovine serum albumin+2 mM EDTA) and werere-pelleted by centrifugation as described above. Cells werere-suspended in 3 mL 1×RBC Lysis Buffer and incubated at roomtemperature for 2-5 minutes. PBE was added to a final volume of 25 mLand cells were pelleted by centrifugation. Cells were re-suspended in 5mL PBE, passed through a 40 μm nylon mesh filter, and harvested bycentrifugation. Cells were re-suspended in 1 mL PBE. Cell concentrationand viability were determined using trypan blue exclusion on a TC-20cell counter (Biorad) prior to preparation for PARylation analysis.Following cell count, 1.5 mL bone marrow cells were pelleted bycentrifugation at 1200 rpm (300 rcf) for 5 minutes at 4° C. Supernatantwas collected and stored at −80° C. for possible analysis of drugconcentration. The remaining 2.5 mL of cells were pelleted bycentrifugation at 1200 rpm (300 rcf) for 5 minutes at 4° C. Pelletedcells were lysed in RIPA buffer containing protease and phosphataseinhibitors (1004, buffer per 10⁶ cells) and stored at −20° C. formeasurement of PARylation in bone marrow cells as determined by an ElisaPARylation assay.

PARylation in Bone Marrow Cells (R⁸H-15, Example 12)

Bone marrow cells isolated from femurs on Day 3 were lysed in RIPAbuffer containing protease/phosphatase inhibitors. The homogenizedsamples were brought to 1% SDS final concentration, heated at 100° C.for 5 minutes, quenched on ice and clarified by centrifugation at12,000×g for 5 minutes at 4° C. PARylation analysis was performed usingTrevigen's HT PARP in vivo Pharmacodynamic Assay II. Briefly, duplicate10 μg samples were loaded on pre-coated/pre-blocked ELISA strip wells as50 μL volumes in sample buffer and incubated for 16 hours at 4° C. intandem with serially diluted purified PAR standards. The wells werewashed 4× with PBST and incubated with 50 μL/well of PAR polyclonaldetecting antibody in antibody diluent for 2 hours at room temperature.The wells were washed 4× with PBST and incubated with 50 μL/well goatanti-rabbit IgG-HRP conjugate in antibody diluent for 1 hour at roomtemperature. The wells were washed 4× with PBST, incubated with 100m/well of 1:1 PARP PeroxyGlow A and PARP PeroxyGlow B and read in aBioTek Cytation 5 using the luminescence fiber endpoint. Data wereplotted as PAR (pg/mL) using values calculated from the standard curve.

Bone Marrow Collection (R⁸H-16, Example 18)

Following tumor collection, femurs were removed, and bone marrow wasextruded into 50 mL conical tubes by flushing the bones with a 23-gaugeneedle fitted on a 5 cc syringe containing PBS+2% fetal bovine serum(FBS). Bone marrow was homogenized by gentle pipetting and filteredthrough 100 μm nylon mesh filters and cells were pelleted bycentrifugation at 1200 rpm for 5 minutes at 4° C. Red blood cells werelysed with 3 mL of lysis buffer for 2 minutes at room temperature. PBSwas added to a volume of 25 mL and cells were re-pelleted bycentrifugation as described above. Cell pellets were suspended in 5 mLof PBS and cell count was assessed by trypan blue exclusion on a TC-20cell counter (BioRad). A subset of 2.5×10⁶ cells from each sample wascollected into a microfuge tube for measurement of PARylation.

PARylation in Bone Marrow Cells (R⁸H-16, Example 18)

Bone marrow cells (2.5×10⁶ cells per pellet) were lysed in 500 μL ofRIPA/HALT buffer and incubated on ice for 15 minutes with periodicvortexing. Cell lysates were brought up to a 1% SDS final concentration,heated at 100° C. for 5 minutes and quenched on ice. Cellular debris wasremoved by centrifugation at 14,000×g for 5 minutes 4° C. and pelletedcells were resuspended in buffer at a concentration of 250,000 cells/50μL. Bone marrow cell samples were loaded on pre-coated/pre-blocked ELISAstrip wells and incubated for 16 hours at 4° C. Samples were washed 4×with PBST (1 L PBS+1 mL Tween 20) and incubated with 50 μL PAR detectingantibody for 2 hours at room temperature. Samples were washed 4× withPBST and incubated with 50 μL goat anti-rabbit IgG-HRP conjugate for 1hour at room temperature. Following a final 4× wash with PBST, 1004, ofPeroxyGlow™ was added to each sample and luminescence was determined ona BioTek Cytation 5 using luminescence fiber endpoint and strip wellarea plate definition.

Statistical Analysis

Analysis of variance (ANOVA) was used to test for significantdifferences between groups. Post-hoc Bonferroni multiple comparison testanalysis was used to determine significant differences among means. Allstatistical analysis was accomplished using Graph Pad Prism 7.03software.

FIG. 5 shows the PARylation in bone marrow cells following intravenousadministration of Example 12 or oral administration of R⁸H-15 incombination with oral administration of temozolomide (TMZ) to nude mice.

FIG. 6 shows PARylation in bone marrow cells following intraperitonealadministration of Example 18 or R⁸H-16 to nude mice.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including withoutlimitation all patent, patent applications, and publications, cited inthe present application is incorporated herein by reference in itsentirety.

What is claimed is:
 1. A compound of formula (I)R8-Q-R7   (I) or a pharmaceutically acceptable salt thereof, wherein: R⁷is a peptide; R⁸ is selected from the group consisting of:

Q is selected from the group consisting of

R¹, R², R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹² are each independentlyselected from H, C₁₋₄ alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1), wherein said C₁₋₄ alkyl, C₁₋₄ alkenyl, C₆₋₁₀aryl, and 5-10 membered heteroaryl are each optionally substituted with1, 2, or 3 substituents independently selected from halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O) NR^(c1)R^(d1); or R¹ and R² togetherwith the carbon atom to which they are attached form a C3-7 cycloalkylgroup optionally substituted with 1, 2, or 3 substituents independentlyselected from halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1); or R¹ and R³ together with the carbon atom towhich they are attached form a C₃₋₇ cycloalkyl group optionallysubstituted with 1, 2, or 3 substituents independently selected fromhalo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1),C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1),NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1); orR³ and R⁴ together with the carbon atom to which they are attached forman C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1); or R⁵ and R⁶ togetherwith the carbon atom to which they are attached form an C₃₋₇ cycloalkylgroup optionally substituted with 1, 2, or 3 substituents independentlyselected from halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1); R¹³ is H or C₁₋₆ alkyl; A is H or C₁₋₄ alkyl;

is C₆₋₁₀ aryl or 5-10 membered heteroaryl; wherein the 5-10 memberedheteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4ring-forming heteroatoms independently selected from N, O, and S; [N, O,S] is NH, O, or S; [N, O] is NH or O; [C, N, O] is CR^(X)R^(Y), NH, orO; each R^(X) and R^(Y) is independently selected from H and C₁₋₄ alkyl;[AA]_(X) is a peptide that may be cleaved by enzymatic action; S1 is

each R^(a), R^(b), R^(c), and R^(d) is independently selected from H,C₁₋₄ alkyl, OR^(a2), CO₂R^(a2), and OC(═O)R^(a2), wherein said C₁₋₄alkyl is optionally substituted with OR^(a2), CO₂R^(a2), andOC(═O)R^(a2); R^(a1), R^(b1), R^(c1), and R^(c1) are each independentlyselected from H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl,OH, CN, NO₂, and CO₂CH₃; wherein said C₁₋₆ alkyl and C₂₋₆ alkenyl areeach optionally substituted with OH, CN, NO₂, or CO₂CH₃; R^(a2) is H orC1-4 alkyl; and n is 0 or
 1. 2. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R⁷ is a peptidecapable of selectively delivering 10-across a cell membrane having anacidic or hypoxic mantle having a pH less than about 6.0.
 3. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R⁷ is a peptide comprising at least one of the followingsequences: (SEQ ID NO: 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG;(SEQ ID NO: 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG;(SEQ ID NO: 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG;(SEQ ID NO: 4; Pv4) Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG; and(SEQ ID No. 5; Pv5) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC;

and wherein R⁷ is attached to Q through a cysteine residue of R⁷.
 4. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R⁷ is a peptide comprising at least one of the followingsequences: (SEQ ID NO: 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG,(SEQ ID NO: 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, and(SEQ ID NO: 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG,

and wherein R⁷ is attached to Q through a cysteine residue of R⁷.
 5. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R⁷ is a peptide comprising the sequence: (SEQ ID NO: 1; Pv1)ADDQNPWRAYLDLLFPTDTLLLDLLWCG.


6. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R⁷ is a peptide comprising the sequence:(SEQ ID NO: 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.


7. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R⁷ is a peptide comprising the sequence:(SEQ ID NO: 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG.


8. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R⁷ is a peptide comprising the sequence:(SEQ ID NO: 4; Pv4) Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG.


9. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R⁷ is a peptide comprising the sequence:(SEQ ID No. 5; Pv5) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC.


10. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ and R² are each independently selected from H andmethyl, and R³, R⁴, R⁵, and R⁶ are each H.
 11. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein R¹ and R² areeach independently selected from H and methyl.
 12. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein R¹ and R² areeach H.
 13. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein R³ and R⁴ are each H.
 14. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein R⁵ and R⁶ areeach H.
 15. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein R⁹, R¹⁰, R¹¹, and R¹² are each independentlyselected from H and methyl.
 16. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein the enzyme capable ofcleaving [AA]_(X) is Cathepsin B, MMPXX, DPPIV, glycoprotein, peptidase,or caspase.
 17. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein [AA]_(X) is a peptide having two to tenamino acid residues.
 18. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein S1 is a group having the followingstructure:


19. A pharmaceutical composition that comprises a compound of claim 1,or a pharmaceutically acceptable salt thereof.
 20. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein R8 is:


21. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein Q is:


22. The compound of claim 21, or a pharmaceutically acceptable saltthereof, wherein R¹ and R² are each independently selected from H andmethyl.
 23. The compound of claim 21, or a pharmaceutically acceptablesalt thereof, wherein R³ and R⁴ are each H.
 24. A compound, which is:

or a pharmaceutically acceptable salt thereof; wherein Pv1 is a peptidecomprising the following sequence: ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ IDNO: 1).
 25. A compound, which is:

or a pharmaceutically acceptable salt thereof; wherein Pv1 is a peptidecomprising the following sequence: (SEQ ID NO: 1)ADDQNPWRAYLDLLFPTDTLLLDLLWCG.


26. A compound, which is:

or a pharmaceutically acceptable salt thereof; wherein Pv2 is a peptidecomprising the following sequence: (SEQ ID NO: 2)AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.


27. A pharmaceutical composition comprising a compound of claim 24, or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable carrier.
 28. A pharmaceutical compositioncomprising a compound of claim 25, or a pharmaceutically acceptable saltthereof, and at least one pharmaceutically acceptable carrier.
 29. Apharmaceutical composition comprising a compound of claim 26, or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable carrier.